Compositions and methods for treating or preventing conditions and diseases associated with mannheimia haemolytica

ABSTRACT

Particular aspects show that the signal peptide remains intact on the mature CD18 molecule on ruminant leukocytes rendering these cells susceptible to cytolysis by Lkt. Comparative amino acid sequence analysis of the signal peptide of CD18 of eight ruminants and five non-ruminants revealed that the ruminant CD18 signal peptides contain ‘cleavage-inhibiting’ glutamine (Q), compared to ‘cleavage-conducive’ glycine in non-ruminants, at position −5 relative to the cleavage site. Mutagenesis of Q at position −5 of the bovine CD18 signal peptide to G resulted in the abrogation of Lkt-mediated cytolysis of transfectants expressing bovine CD18 carrying the Q(−5)G mutation. Provided is novel technology to clone cattle and other ruminants expressing CD18 without the signal peptide on their leukocytes, providing ruminants that are less susceptible to  M. haemolytica . Methods for treating conditions and/or diseases associated with  M. haemolytica  (e.g., pneumonic pasteurellosis), comprising administration of polypeptides comprising CD18 signal peptide sequences are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority to U.S. ProvisionalPatent Application Ser. No. 61/149,278 filed 2 Feb. 2009 and entitledCOMPOSITIONS AND METHODS FOR TREATING OR PREVENTING CONDITIONS ANDDISEASES ASSOCIATED WITH MANNHEIMIA HAEMOLYTICA, which is incorporatedby reference herein in its entirety.

FIELD OF THE INVENTION

Aspects of the present invention relate generally to conditions and/ordiseases associated with M. haemolytica in ruminants, and moreparticularly to novel and efficacious compositions and methods fortreating or preventing conditions and/or diseases associated with M.haemolytica in ruminants.

BACKGROUND

Mannheimia haemolytica (M. haemolytica) is the most significantbacterial pathogen of respiratory disease in cattle, sheep, goats andother ruminants, and causes extensive economic losses world-wide⁸ . M.haemolytica is commonly found in the nasopharynx of healthy ruminants.In conjunction with active viral infection and stress factors, M.haemolytica migrates to the lungs, where it multiplies rapidly, andcauses a fibrinonecrotic pleuropneumonia. M. haemolytica producesseveral virulence factors⁸. Based on the observation thatleukotoxin-deletion mutants (Lkt-deletion mutants) of M. haemolyticacause reduced mortality and much milder lung lesions than the wild-typeorganisms, Lkt is considered as the most important virulence factor ofthis organism⁹⁻¹³. Lkt belongs to the family of RTX (repeats in toxins)toxins, and shares extensive homology with the exotoxins produced byother gram-negative bacteria such as Escherichia coli ¹⁴ ,Actinobacillus pleuropneumoniae ¹⁵, and Actinobacillusactinomycetemcomitans ¹⁶. Cytolytic activity of M. haemolytica Lkt isspecific for ruminant leukocytes¹⁷⁻¹⁸. Although all the subsets ofleukocytes are susceptible to the cytolytic effects of Lkt, PMNs are themost susceptible subset¹⁹. PMN-depletion mitigates the lung injury incalves caused by M. haemolytica infection²⁰. Therefore, Lkt-induced PMNlysis and degranulation are the primary causes of acute inflammation andlung injury characteristic of pneumonic pasteurellosis^(8,13,20).

There is, therefore a pronounced need in the art for novel andefficacious compositions and methods for treating or preventingconditions and/or diseases associated with M. haemolytica (e.g., inmammals, ruminants).

SUMMARY OF EXEMPLARY ASPECTS OF THE INVENTION

In particular aspects of the present invention, studies aimed at mappingthe Mannheimia (Pasteurella) haemolytica leukotoxin (Lkt) binding siteon its receptor CD18 have unexpectedly shown, as disclosed herein, thatthe signal peptide of ruminant CD18 remains intact on the mature CD18molecule on the leukocytes of ruminants and renders these cellssusceptible to cytolysis by Lkt.

In additional aspects, comparative analysis of the amino acid (aa)sequence of the signal peptide of CD18 of eight ruminants and fivenon-ruminants revealed that the signal peptide of CD18 of ruminantscontain ‘cleavage-inhibiting’ glutamine (Q), whereas that ofnon-ruminants contain ‘cleavage-conducive’ glycine (G) at position −5relative to the cleavage site.

In further aspects, site-directed mutagenesis of Q at position −5 of thesignal peptide of bovine CD18 to G resulted in the abrogation ofcytolysis of transfectants expressing bovine CD18 carrying the Q(−5)Gmutation.

Particular aspects, therefore provide a hitherto unavailable technologyto clone cattle and other ruminants expressing CD18 without the signalpeptide on their leukocytes, providing ruminants that are lesssusceptible to pneumonic pasteurellosis that costs millions of dollarsworld-wide annually.

Particular preferred aspects provide a purified or recombinant ruminantCD18 polypeptide, comprising a ruminant CD18 polypeptide, or portionthereof, having a cleavable signal peptide with a helix-breaking aminoacid residue at amino acid position 18 (−5 with respect to signalpeptide cleavage site). In certain aspects, the amino acid residue atamino acid position 18 is selected from the group consisting of glycine,proline, arginine, and tyrosine. In further preferred aspects, theruminant is selected from the group consisting of cattle, bison,buffalo, goat, domestic sheep, big horn sheep, deer, elk, giraffes,yaks, camels, alpacas, llamas, wildebeest, antelope, pronghorn andnilgai. Yet further aspects provide, that the recombinant ruminant CD18polypeptide is one Q(−5)G CD18 mutant selected from the group consistingof SEQ ID NOS:57, 58, 60, 62, 64, 66, 68, 70 and CD18 signalpeptide-comprising portions thereof.

Additional aspects provide an isolated nucleic acid comprising asequence that encodes the polypeptide of CD18 having a cleavable signalpeptide with a helix breaking amino acid residue at amino acid position18.

Further preferred aspects provide for a recombinant expression vector,comprising a nucleic acid comprising a sequence that encodes apolypeptide comprising a ruminant CD18 polypeptide, or portion thereof,having a cleavable signal peptide with a helix-breaking amino acidresidue at amino acid position 18 (−5 with respect to signal peptidecleavage site).

Yet further preferred aspects provide for a recombinant or clonedruminant cell or ruminant animal, comprising a ruminant cell capable ofexpressing a polypeptide comprising a ruminant CD18 polypeptide, orportion thereof, having a cleavable signal peptide with a helix-breakingamino acid residue at amino acid position 18 (−5 with respect to signalpeptide cleavage site). Additional aspects provide that expression of apolypeptide comprises a ruminant CD18 polypeptide having a cleavablesignal peptide comprises expressing from a genomic locus, or from arecombinant expression vector. Further aspects provide that the cell oranimal is less susceptible to, or resistant to the effects of M.haemolytica, relative to wild-type control cells.

Additional aspects provide that the cell or animal is that of a ruminantselected from the group consisting of cattle, bison, buffalo, goat,domestic sheep, big horn sheep, deer, elk, giraffes, yaks, camels,alpacas, llamas, wildebeest, antelope, pronghorn and nilgai. Furtherpreferred aspects provide that the cell capable of expressing apolypeptide comprising a ruminant CD18 polypeptide, or portion thereof,having a cleavable signal peptide, there is reduced or no expression ofthe endogenous wild-type CD18 polypeptide having a non-cleavable signalpeptide.

Further preferred aspects provide for a method of providing arecombinant or cloned ruminant cell, comprising introduction into, orengineering within the ruminant cell, a nucleic acid comprising asequence that encodes a polypeptide comprising a ruminant CD18polypeptide, or portion thereof, having a cleavable signal peptide witha helix-breaking amino acid residue at amino acid position 18 (−5 withrespect to signal peptide cleavage site), wherein the cell is lesssusceptible to, or resistant to the effects of M. haemolytica, relativeto wild-type control cells. Yet further aspects provide for the aminoacid residue at amino acid position 18 is selected from the groupconsisting of glycine, proline, arginine, and tyrosine.

Additional preferred aspects provide for a method where the ruminant isselected from the group consisting of cattle, bison, buffalo, goat,domestic sheep, big horn sheep, deer, elk, giraffes, yaks, camels,alpacas, llamas, wildebeest, antelope, pronghorn and nilgai. Furtherpreferred aspects provide for the recombinant ruminant CD18 polypeptideis one Q(−5)G CD18 mutant selected from the group consisting of SEQ IDNOS:57, 58, 60, 62, 64, 66, 68, 70 and CD18 signal peptide-comprisingportions thereof.

Further preferred aspects provide a method of providing a recombinant orcloned ruminant animal, comprising introduction into, or engineeringwithin one or more cells of a ruminant animal, a nucleic acid comprisinga sequence that encodes a polypeptide comprising a ruminant CD18polypeptide, or portion thereof, having a cleavable signal peptide witha helix-breaking amino acid residue at amino acid position 18 (−5 withrespect to signal peptide cleavage site), wherein the recombinant orcloned ruminant animal is less susceptible to, or resistant to theeffects of M. haemolytica, relative to wild-type control cells. Yetfurther aspects provide for the use of ruminant stem cells or enucleatedruminant cells.

Additional preferred aspects provide a method where the amino acidresidue at amino acid position 18 is selected from the group consistingof glycine, proline, arginine, and tyrosine. Further preferred aspectsprovide a method where the ruminant is selected from the groupconsisting of cattle, bison, buffalo, goat, domestic sheep, big hornsheep, deer, elk, giraffes, yaks, camels, alpacas, llamas, wildebeest,antelope, pronghorn and nilgai.

Further preferred aspects provide a method where the recombinantruminant CD18 polypeptide is one Q(−5)G CD18 mutant selected from thegroup consisting of SEQ ID NOS:57, 58, 60, 62, 64, 66, 68, 70 and CD18signal peptide-comprising portions thereof.

Yet further preferred aspects provide a method of treating or preventingconditions and diseases associated with M. haemolytica in ruminants,comprising administering to a ruminant subject in need thereof, anamount of a polypeptide comprising a CD18 signal peptide, or portionthereof, suitable to treat, prevent or otherwise ameliorate a conditionor diseases associated with M. haemolytica in the ruminant.

Additional aspects provide a method of treating whereby the polypeptidecomprising a CD18 signal peptide, or portion thereof, is a polypeptidecomprising from about 13 to about 24 contiguous amino acid residues ofthe first 24 amino acids of the N-terminus of the native (full-lengthnascent) CD18 sequence, wherein the polypeptide is suitable to providefor at least one of binding to M. haemolytica leukotoxin (Lkt) andabrogation of Lkt-induced cytolysis. Further aspects provide a methodwhereby the polypeptide comprises a contiguous portion of the CD18signal polypeptide beginning at amino acid residue 5. Yet furtheraspects provide a method whereby the polypeptide comprises residues 5 to17 of the CD18 signal polypeptide.

Further preferred aspects provide a method whereby the CD18 sequence isselected from the group consisting of cattle, bison, buffalo, goat,domestic sheep, big horn sheep, deer, elk, giraffes, yaks, camels,alpacas, llamas, wildebeest, antelope, pronghorn, nilgai, human, chimp,mouse, rat and pig. Additional preferred aspects provide a methodwhereby the CD18 sequence is selected from the group consisting of SEQID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 21, 26, 57, 58, 60, 62,64, 66, 68 and 70.

Certain preferred aspects provide a method whereby the CD18 signalpeptide sequence is selected from the group consisting of cattle, bison,buffalo, goat, domestic sheep, big horn sheep, deer, elk, giraffes,yaks, camels, alpacas, llamas, wildebeest, antelope, pronghorn andnilgai, human, chimp, mouse, rat and pig. Additional preferred aspectsprovide a method whereby the CD18 signal peptide sequence is selectedfrom the group consisting of SEQ ID NOS:28, 30, 32, 34, 36, 40, 42, 44,46, 48, 50, 52 and 54.

Certain aspects further comprise administration of an anti-leukotoxinantibody reagent or epitope-binding portion thereof.

In particular aspects, administering the amount of the polypeptidecomprising a CD18 signal peptide, or portion thereof, comprisesadministration to a ruminant previously vaccinated against M.haemolytica. In certain embodiments, vaccination against M. haemolyticacomprises administration of M. haemolytica leukotoxin (Lkt) or a portionthereof.

Additional aspects provide an antibody specific for a CD18 signalpeptide, or portion thereof comprising from about 13 to about 24contiguous amino acid residues of the first 24 amino acids of theN-terminus of the native (full-length nascent) ruminant CD18 sequence,wherein the polypeptide is suitable to provide for at least one ofbinding to M. haemolytica leukotoxin (Lkt) and abrogation of Lkt-inducedcytolysis. In certain embodiments, the polypeptide comprises acontiguous portion of the CD18 signal polypeptide beginning at aminoacid residue 5. In certain aspects, the polypeptide comprises residues 5to 17 of the CD18 signal polypeptide. In particular embodiments, theCD18 sequence is selected from the group consisting of cattle, bison,buffalo, goat, domestic sheep, big horn sheep, deer, elk, giraffes,yaks, camels, alpacas, llamas, wildebeest, antelope, pronghorn, nilgai,human, chimp, mouse, rat and pig. In certain embodiments, the CD18sequence is selected from the group consisting of SEQ ID NOS:2, 4, 6, 8,10, 12, 14, 16, 18, 20, 22, 21, 26, 57, 58, 60, 62, 64, 66, 68 and 70.

Yet further aspects provide a method of treating for treating orpreventing conditions and diseases associated with M. haemolytica inruminants, comprising administering to a ruminant subject in needthereof, an amount of an antibody specific for a CD18 signal peptide, orportion thereof comprising from about 13 to about 24 contiguous aminoacid residues of the first 24 amino acids of the N-terminus of thenative (full-length nascent) ruminant CD18 sequence, wherein thepolypeptide is suitable to provide for at least one of binding to M.haemolytica leukotoxin (Lkt) and abrogation of Lkt-induced cytolysis,wherein a method of treating for treating or preventing conditions anddiseases associated with M. haemolytica in ruminants is provided. Incertain aspects, the antibody is according to those anti-ruminant CD18signal peptide antibodies described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C show, according to particular exemplary aspects, that theCD18 signal peptide analog P5 (aa 5-24) inhibits Lkt-induced cytolysisof bovine PMNs. See working EXAMPLE 2 for details.

FIGS. 2A and 2B show, according to particular exemplary aspects, thatpeptide P5 inhibits Lkt binding to bovine PMNs. See working EXAMPLE 2for details.

FIGS. 3A-3C show, according to particular exemplary aspects, that N- andC-terminal truncations of peptide P5 identifies the minimal peptidesequence of bovine CD18 bound by Lkt as aa 5-17. See working EXAMPLE 2for details.

FIG. 4A-4H show, according to particular exemplary aspects, thatanti-signal peptide serum binds to membrane CD18 of PMNs of allruminants tested. See working EXAMPLE 3 for details.

FIGS. 5A-5D show, according to particular exemplary aspects, that thesignal peptide of bovine CD18 is not cleaved. See working EXAMPLE 4 fordetails.

FIGS. 6A-6B show, according to particular exemplary aspects, Signalpeptide of CD18 of ruminants contain glutamine at aa position −5relative to the cleavage site, whereas that of non-ruminants containglycine. See working EXAMPLE 5 for details.

FIG. 7 shows, according to particular exemplary aspects, that mutationof glutamine at position −5 of the signal peptide of bovine CD18 toglycine abrogates Lkt-induced cytolysis of transfectants expressing CD18with Q(−5)G mutation. See working EXAMPLE 6 for details.

FIGS. 8A-8C shows, according to particular exemplary aspects, thatpeptides spanning aa 500-600 of bovine CD18 fail to inhibit Lkt-inducedcytolysis of bovine PMNs. See working EXAMPLE 6 for details.

FIG. 9 shows, according to particular exemplary aspects, thatco-incubation of peptide P17 with in vitro cultures of M. haemolyticaabrogate leukotoxic activity. See working EXAMPLE 7 for details.

FIG. 10 shows mean clinical scores of calves inoculated with M.haemolytica only (Group I), M. haemolytica along with the irrelevantpeptide PSC (Group II) and M. haemolytica along with the peptide P17(Group III) at different time points post-inoculation. At 24 hourspost-inoculation, the mean clinical score of Group III was statisticallysignificantly lower than that of other groups (P<0.05). See workingEXAMPLE 7 for details.

FIGS. 11A-11B shows, according to particular aspects, representativegross-(A) and histo-(B) pathology of the lungs of calves infected withM. haemolytica with or without peptides. See working EXAMPLE 7 fordetails.

DETAILED DESCRIPTION OF EXEMPLARY ASPECTS OF THE INVENTION

Generally, a nascent membrane protein contains a signal sequence thatdirects the protein/ribosome to the endoplasmic reticulum (ER)membrane¹⁻³. The signal peptide binds to the signal recognition particle(SRP) which in turn binds to the SRP receptor on the ER membrane andhelps in the translocation of the protein into the lumen of the ER. Thesignal peptide is cleaved from the protein by the ER-resident signalpeptidase while it is still growing on the ribosome. Thus the signalpeptide is not present on the mature protein that reaches the plasmamembrane following post-translational modifications.

As disclosed herein, however, Applicants' mapping the Mannheimia(Pasteurella) haemolytica leukotoxin (Lkt) binding site on its receptorCD18 have led to the unexpected finding that the signal peptide ofruminant CD18 remains intact on the mature CD18 molecule on theleukocytes of ruminants and renders these cells susceptible to cytolysisby Lkt.

Therefore, the signal peptide of ruminant CD18, the β subunit ofleukocyte-specific β2-integrins, is an exception to general phenomenonthat signal peptides are not present on the mature protein that reachesthe plasma membrane. Intriguingly, as disclosed herein, the intactsignal peptide of CD18 is responsible for the susceptibility of ruminantleukocytes to Mannheimia (Pasteurella) haemolytica leukotoxin, and theresultant susceptibility of ruminants to severe pneumonia caused by thisorganism.

Previously, Applicants identified CD18 as the receptor for Lkt onbovine⁴ and ovine^(5,6) leukocytes, and mapped the Lkt-binding site tolie between amino acids 1-291⁷. As disclosed herein, under workingEXAMPLE 2, inhibition of Lkt-induced cytolysis of ruminant leukocytes byCD18 peptide analogs revealed that the Lkt-binding site is formed by aa5-17 of CD18 which, surprisingly, comprise a part of the signal peptide.

As shown herein under working EXAMPLE 3, flow cytometric analysis ofruminant leukocytes with an anti-signal peptide serum indicated thepresence of the signal peptide on the mature CD18 molecules expressed onthe cell surface.

As shown herein under working EXAMPLE 4, analysis of the transfectantsexpressing CD18 containing the ‘FLAG’ epitope at the putative cleavagesite confirmed that the signal peptide of CD18 is not cleaved.

Working EXAMPLE 5 below, discloses a comparative analysis of the aminoacid (aa) sequence of the signal peptide of CD18 of eight ruminants andfive non-ruminants, and revealed that the signal peptide of CD18 ofruminants contain ‘cleavage-inhibiting’ glutamine (Q), whereas that ofnon-ruminants contain ‘cleavage-conducive’ glycine (G) at position −5relative to the cleavage site.

Working EXAMPLE 6 below, discloses that site-directed mutagenesis of Qat position −5 of the signal peptide of bovine CD18 to G resulted in theabrogation of cytolysis of transfectants expressing bovine CD18 carryingthe Q(−5)G mutation. According to particular aspects, replacement of‘cleavage-inhibiting’ Q at −5 position with ‘cleavage-conducive’ Gresulted in the cleavage of the signal peptide and the resultant loss ofsusceptibility of the transfectants to Lkt-induced cytolysis. Accordingto additional aspects, it is possible that abrogation of cytolysis isnot due to cleavage of signal peptide, but due to conformational changescaused by the replacement of Q with G. Irrespective of the molecularbasis underlying the abrogation of cytolysis, however, the exemplaryQ(−5)G mutation presents an exemplary embodiment of a hithertounavailable technology to, among other things, clone cattle and otherruminants expressing CD18 without the signal peptide on theirleukocytes, and thus provide animals that are substantially lesssusceptible to pneumonic pasteurellosis.

Definitions: “Functional variants” as used herein refers to at least oneprotein selected from the group consisting of SEQ ID NOS:2, 4, 6, 8, 10,12 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46,48, 50, 52, 54, 57, 58, 60, 62, 64, 66, 68, and 70 sequences having atleast 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%sequence identity thereto, and biologically active variants thereof,where functional or biologically active variants are those proteins thatdisplay one or more of the biological activities of at least one proteinselected from the group consisting of SEQ ID NOS: 2, 4, 6, 8, 10, 12 14,16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50,52, 54, 57, 58, 60, 62, 64, 66, 68, and 70, sequences having at least60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequenceidentity thereto, including but not limited to the activities disclosedherein (e.g., binding to the bacterial toxin Lkt; producing proteinsresistant to binding by Lkt)

As used herein, a pharmaceutical or therapeutic effect refers to aneffect observed upon administration of an agent intended for theprevention or treatment of a disease or disorder or for amelioration ofthe symptoms thereof.

As used herein, treatment means any manner in which the symptoms of acondition, disorder or disease or other indication, are ameliorated orotherwise beneficially altered.

As used herein therapeutic effect means an effect resulting fromtreatment of a subject that alters, typically improves or amelioratesthe symptoms of a disease or condition or that cures a disease orcondition. A therapeutically effective amount refers to the amount of acomposition, molecule or compound which results in a therapeutic effectfollowing administration to a subject.

As used herein, the term “subject” refers to ruminant animals, includingmammals, such as cattle.

As used herein, the phrase “associated with” refers to certainbiological aspects such as expression of a receptor or signaling by areceptor that occurs in the context of a disease or condition. Suchbiological aspect may or may not be causative or integral to the diseaseor condition but merely an aspect of the disease or condition.

As used herein, a biological activity refers to a function of apolypeptide including but not limited to complexation, dimerization,multimerization, receptor-associated kinase activity,receptor-associated protease activity, phosphorylation,dephosphorylation, autophosphorylation, ability to form complexes withother molecules, ligand binding, catalytic or enzymatic activity,activation including auto-activation and activation of otherpolypeptides, inhibition or modulation of another molecule's function,stimulation or inhibition of signal transduction and/or cellularresponses such as cell proliferation, migration, differentiation, andgrowth, degradation, membrane localization, membrane binding, andoncogenesis. A biological activity can be assessed by assays describedherein and by any suitable assays known to those of skill in the art,including, but not limited to in vitro assays, including cell-basedassays, in vivo assays, including assays in animal models for particulardiseases.

Table 1 contains a brief description and sequence listing including somebut not all (e.g., exemplary) of the peptides and polypeptides withinthe scope of this invention. Therapeutic peptides to be used in theprevention and/or treatment of an infection of M. haemolytica may be aportion of CD18 from ruminants containing the signal sequence. Inpreferred aspects, the therapeutic contains the amino acid residues ofCD18 from ruminants from amino acids 1 to 25, 1 to 24, 1 to 23, 1 to 22,1 to 21, 1 to 20, 1 to 19, 1 to 18, 1 to 17, 1 to 16, 1 to 15, 1 to 14,1 to 13, 1 to 12, 1 to 11, and 1 to 10. Additionally, the therapeuticcontains the amino acid residues of CD18 from ruminants from amino acids2 to 25, 2 to 24, 2 to 23, 2 to 22, 2 to 21, 2 to 20, 2 to 19, 2 to 18,2 to 17, 2 to 16, 2 to 15, 2 to 14, 2 to 13, 2 to 12, 2 to 11, and 2 to10. In further preferred embodiments, the therapeutic contains the aminoacid residues of CD18 from ruminants from amino acids 3 to 25, 3 to 24,3 to 23, 3 to 22, 3 to 21, 3 to 20, 3 to 19, 3 to 18, 3 to 17, 3 to 16,3 to 15, and 3 to 14, 3 to 13, 3 to 12, 3 to 11, and 3 to 10. In yetfurther preferred embodiments, the therapeutic contains the amino acidresidues of CD18 from ruminants from amino acids 4 to 25, 4 to 24, 4 to23, 4 to 22, 4 to 21, 4 to 20, 4 to 19, 4 to 18, 4 to 17, 4 to 16, 4 to15, and 4 to 14, 4 to 13, 4 to 12, 4 to 11, and 4 to 10. In additional,preferred embodiments, the therapeutic contains the amino acid residuesof CD18 from ruminants from amino acids 5 to 25, 5 to 24, 5 to 23, 5 to22, 5 to 21, 5 to 20, 5 to 19, 5 to 18, 5 to 17, 5 to 16, 5 to 15, and 5to 14, 5 to 13, 5 to 12, 5 to 11, and 5 to 10.

TABLE 1 Summary of exemplary SEQ ID NOS and brief descriptions thereof.SEQ ID NO BRIEF DESCRIPTION Nucleic Acid Protein Full Length CD18Protein Cattle SEQ ID NO: 1 SEQ ID NO: 2 Bison SEQ ID NO: 3 SEQ ID NO: 4Buffalo SEQ ID NO: 5 SEQ ID NO: 6 Goat SEQ ID NO: 7 SEQ ID NO: 8Domestic Sheep SEQ ID NO: 9 SEQ ID NO: 10 Wild Sheep SEQ ID NO: 11 SEQID NO: 12 Deer SEQ ID NO: 13 SEQ ID NO: 14 Elk SEQ ID NO: 15 SEQ ID NO:16 Human SEQ ID NO: 17 SEQ ID NO: 18 Mouse SEQ ID NO: 19 SEQ ID NO: 20Rat SEQ ID NO: 21 SEQ ID NO: 22 Pig SEQ ID NO: 23 SEQ ID NO: 24 ChimpSEQ ID NO: 25 SEQ ID NO: 26 Exemplary Mutated CD18 Cattle SEQ ID NO: 55SEQ ID NO: 58 Bison SEQ ID NO: 59 SEQ ID NO: 60 Buffalo SEQ ID NO: 56SEQ ID NO: 57 Goat SEQ ID NO: 61 SEQ ID NO: 62 Domestic Sheep SEQ ID NO:63 SEQ ID NO: 64 Wild Sheep SEQ ID NO: 65 SEQ ID NO: 66 Deer SEQ ID NO:67 SEQ ID NO: 68 Elk SEQ ID NO: 69 SEQ ID NO: 70 Peptide from CD18Signal Sequence Cattle SEQ ID NO: 27 SEQ ID NO: 28 Bison SEQ ID NO: 27SEQ ID NO: 28 Buffalo SEQ ID NO: 29 SEQ ID NO: 30 Goat SEQ ID NO: 31 SEQID NO: 32 Domestic Sheep SEQ ID NO: 33 SEQ ID NO: 34 Wild Sheep SEQ IDNO: 33 SEQ ID NO: 34 Deer SEQ ID NO: 35 SEQ ID NO: 36 Elk SEQ ID NO: 37SEQ ID NO: 38 Human SEQ ID NO: 39 SEQ ID NO: 40 Chimp SEQ ID NO: 39 SEQID NO: 40 Mouse SEQ ID NO: 41 SEQ ID NO: 42 Rat SEQ ID NO: 43 SEQ ID NO:44 Pig SEQ ID NO: 45 SEQ ID NO: 46 Exemplary Mutated Peptide from CD18Signal Sequence Human SEQ ID NO: 47 SEQ ID NO: 48 Chimp SEQ ID NO: 47SEQ ID NO: 48 Mouse SEQ ID NO: 49 SEQ ID NO: 50 Rat SEQ ID NO: 51 SEQ IDNO: 52 Pig SEQ ID NO: 53 SEQ ID NO: 54

Variants of at least one protein selected from the group consisting ofSEQ ID NOS:2, 4, 6, 8, 10, 12 14, 16, 18, 20, 22, 24, 26, 28, 30, 32,34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 57, 58, 60, 62, 64, 66, 68,and 70, sequences having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 96%, 97%, 98% or 99% sequence identity thereto have utility foraspects of the present invention. Variants can be naturally ornon-naturally occurring. Naturally occurring variants (e.g.,polymorphisms) are found in humans or other species and comprise aminoacid sequences which are substantially identical to the amino acidsequences disclosed herein. Species homologs of the protein can beobtained using subgenomic polynucleotides of the invention, as describedbelow, to make suitable probes or primers for screening cDNA expressionlibraries from other species, such as mice, monkeys, yeast, or bacteria,identifying cDNAs which encode homologs of the protein, and expressingthe cDNAs as is known in the art.

Non-naturally occurring variants which retain substantially the samebiological activities as naturally occurring protein variants.Preferably, naturally or non-naturally occurring variants have aminoacid sequences which are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 96%, 97%, 98% or 99% identical to the amino acid sequenceddisclosed herein. More preferably, the molecules are at least 98% or 99%identical. Percent identity is determined using any method known in theart. A non-limiting example is the Smith-Waterman homology searchalgorithm using an affine gap search with a gap open penalty of 12 and agap extension penalty of 1. The Smith-Waterman homology search algorithmis taught in Smith and Waterman, Adv. Appl. Math. 2:482-489, 1981.

As used herein, “amino acid residue” refers to an amino acid formed uponchemical digestion (hydrolysis) of a polypeptide at its peptidelinkages. The amino acid residues described herein are generally in the“L” isomeric form. Residues in the “D” isomeric form can be substitutedfor any L-amino acid residue, as long as the desired functional propertyis retained by the polypeptide. NH2 refers to the free amino grouppresent at the amino terminus of a polypeptide. COOH refers to the freecarboxy group present at the carboxyl terminus of a polypeptide. Inkeeping with standard polypeptide nomenclature described in J. Biol.Chem., 243:3552-59 (1969) and adopted at 37 C.F.R. §§1.821-1.822,abbreviations for amino acid residues are shown in Table 2:

TABLE 2 Table of Correspondence SYMBOL 1-Letter 3-Letter AMINO ACID YTyr Tyrosine G Gly Glycine F Phe Phenylalanine M Met Methionine A AlaAlanine S Ser Serine I Ile Isoleucine L Leu Leucine T Thr Threonine VVal Valine P Pro Praline K Lys Lysine H His Histidine Q Gln Glutamine EGlu glutamic acid Z Glx Glu and/or Gln W Trp Tryptophan R Arg Arginine DAsp aspartic acid N Asn Asparagines B Asx Asn and/or Asp C Cys CysteineX Xaa Unknown or other

It should be noted that all amino acid residue sequences representedherein by a formula have a left to right orientation in the conventionaldirection of amino-terminus to carboxyl-terminus. In addition, thephrase “amino acid residue” is defined to include the amino acids listedin the Table of Correspondence and modified and unusual amino acids,such as those referred to in 37 C.F.R. §§1.821-1.822, and incorporatedherein by reference. Furthermore, it should be noted that a dash at thebeginning or end of an amino acid residue sequence indicates a peptidebond to a further sequence of one or more amino acid residues or to anamino-terminal group such as NH) or to a carboxyl-terminal group such asCOOH.

Guidance in determining which amino acid residues can be substituted,inserted, or deleted without abolishing biological or immunologicalactivity can be found using computer programs well known in the art,such as DNASTAR software. Preferably, amino acid changes in the proteinvariants disclosed herein are conservative amino acid changes, i.e.,substitutions of similarly charged or uncharged amino acids. Aconservative amino acid change involves substitution of one of a familyof amino acids which are related in their side chains. Naturallyoccurring amino acids are generally divided into four families: acidic(aspartate, glutamate), basic (lysine, arginine, histidine), non-polar(alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), and uncharged polar (glycine, asparagine,glutamine, cystine, serine, threonine, tyrosine) amino acids.Phenylalanine, tryptophan, and tyrosine are sometimes classified jointlyas aromatic amino acids.

It is reasonable to expect that an isolated replacement of a leucinewith an isoleucine or valine, an aspartate with a glutamate, a threoninewith a serine, or a similar replacement of an amino acid with astructurally related amino acid will not have a major effect on thebiological properties of the resulting variant.

Variants of the at least one protein selected from the group consistingof SEQ ID NOS: 2, 4, 6, 8, 10, 12 14, 16, 18, 20, 22, 24, 26, 28, 30,32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 57, 58, 60, 62, 64, 66,68, and 70, sequences having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 96%, 97%, 98% or 99% sequence identity thereto disclosed hereininclude glycosylated forms, aggregative conjugates with other molecules,and covalent conjugates with unrelated chemical moieties (e.g.,pegylated molecules). Covalent variants can be prepared by linkingfunctionalities to groups which are found in the amino acid chain or atthe N- or C-terminal residue, as is known in the art. Variants alsoinclude allelic variants, species variants, and muteins. Truncations ordeletions of regions which do not affect functional activity of theproteins are also variants.

A subset of mutants, called muteins, is a group of polypeptides in whichneutral amino acids, such as serines, are substituted for cysteineresidues which do not participate in disulfide bonds. These mutants maybe stable over a broader temperature range than native secreted proteins(see, e.g., Mark et al., U.S. Pat. No. 4,959,314).

Preferably, amino acid changes in the variants are conservative aminoacid changes, i.e., substitutions of similarly charged or unchargedamino acids. A conservative amino acid change involves substitution ofone of a family of amino acids which are related in their side chains.Naturally occurring amino acids are generally divided into fourfamilies: acidic (aspartate, glutamate), basic (lysine, arginine,histidine), non-polar (alanine, valine, leucine, isoleucine, proline,phenylalanine, methionine, tryptophan), and uncharged polar (glycine,asparagine, glutamine, cystine, serine, threonine, tyrosine) aminoacids. Phenylalanine, tryptophan, and tyrosine are sometimes classifiedjointly as aromatic amino acids.

It is reasonable to expect that an isolated replacement of a leucinewith an isoleucine or valine, an aspartate with a glutamate, a threoninewith a serine, or a similar replacement of an amino acid with astructurally related amino acid will not have a major effect on thebiological properties of the resulting secreted protein or polypeptidevariant. Properties and functions of the variants are of the same typeas a protein comprising the amino acid sequence encoded by thenucleotide sequence shown in SEQ ID NOS: 2, 4, 6, 8, 10, 12 14, 16, 18,20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54,57, 58, 60, 62, 64, 66, 68, and 70, and sequences having at least 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequenceidentity thereto, although the properties and functions of variants candiffer in degree.

Variants of at least one protein selected from the group consisting ofSEQ ID NOS: 2, 4, 6, 8, 10, 12 14, 16, 18, 20, 22, 24, 26, 28, 30, 32,34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 57, 58, 60, 62, 64, 66, 68,and 70, sequences having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 96%, 97%, 98% or 99% sequence identity thereto include glycosylatedforms, aggregative conjugates with other molecules, and covalentconjugates with unrelated chemical moieties (e.g., pegylated molecules).The variants also include allelic variants, species variants, andmuteins. Truncations or deletions of regions which do not affectfunctional activity of the proteins are also variants. Covalent variantscan be prepared by linking functionalities to groups which are found inthe amino acid chain or at the N- or C-terminal residue, as is known inthe art.

It will be recognized in the art that some amino acid sequences of thepolypeptides of the invention can be varied without significant effecton the structure or function of the protein. If such differences insequence are contemplated, it should be remembered that there arecritical areas on the protein which determine activity. In general, itis possible to replace residues that form the tertiary structure,provided that residues performing a similar function are used. In otherinstances, the type of residue may be completely unimportant if thealteration occurs at a non-critical region of the protein. Thereplacement of amino acids can also change the selectivity of binding tocell surface receptors (Ostade et al., Nature 361:266-268, 1993). Thus,the polypeptides of the present invention may include one or more aminoacid substitutions, deletions or additions, either from naturalmutations or human manipulation.

Of particular interest are substitutions of charged amino acids withanother charged amino acid and with neutral or negatively charged aminoacids. The latter results in proteins with reduced positive charge toimprove the characteristics of the disclosed protein. The prevention ofaggregation is highly desirable. Aggregation of proteins not onlyresults in a loss of activity but can also be problematic when preparingpharmaceutical formulations, because they can be immunogenic (see, e.g.,Pinckard et al., Clin. Exp. Immunol. 2:331-340 (1967); Robbins et al.,Diabetes 36:838-845 (1987); and Cleland et al., Crit. Rev. TherapeuticDrug Carrier Systems 10:307-377 (1993)).

Amino acids in polypeptides of the present invention that are essentialfor function can be identified by methods known in the art, such assite-directed mutagenesis or alanine-scanning mutagenesis (Cunninghamand Wells, Science 244:1081-1085 (1989)). The latter procedureintroduces single alanine mutations at every residue in the molecule.The resulting mutant molecules are then tested for biological activitysuch as binding to a natural or synthetic binding partner. Sites thatare critical for ligand-receptor binding can also be determined bystructural analysis such as crystallization, nuclear magnetic resonanceor photoaffinity labeling (Smith et al., J. Mol. Biol. 224:899-904(1992) and de Vos et al. Science 255:306-312 (1992)).

As indicated, changes are preferably of a minor nature, such asconservative amino acid substitutions that do not significantly affectthe folding or activity of the protein. Of course, the number of aminoacid substitutions a skilled artisan would make depends on many factors,including those described above. Generally speaking, the number ofsubstitutions for any given polypeptide will not be more than 50, 40,30, 25, 20, 15, 10, 5 or 3.

In addition, pegylation of the inventive polypeptides and/or muteins isexpected to provide such improved properties as increased half-life,solubility, and protease resistance. Pegylation is well known in theart.

Fusion Proteins

Fusion proteins comprising proteins or polypeptide fragments of at leastone protein selected from the group consisting of SEQ ID NOS: 2, 4, 6,8, 10, 12 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42,44, 46, 48, 50, 52, 54, 57, 58, 60, 62, 64, 66, 68, and 70, sequenceshaving at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or99% sequence identity thereto can also be constructed. Fusion proteinsare useful for generating antibodies against amino acid sequences andfor use in various targeting and assay systems. For example, fusionproteins can be used to identify proteins which interact with apolypeptide of the invention or which interfere with its biologicalfunction. Physical methods, such as protein affinity chromatography, orlibrary-based assays for protein-protein interactions, such as the yeasttwo-hybrid or phage display systems, can also be used for this purpose.Such methods are well known in the art and can also be used as drugscreens. Fusion proteins comprising a signal sequence can be used.

A fusion protein comprises two protein segments fused together by meansof a peptide bond. Amino acid sequences for use in fusion proteins ofthe invention can be utilize the amino acid sequence shown in SEQ IDNOS: 2, 4, 6, 8, 10, 12 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36,38, 40, 42, 44, 46, 48, 50, 52, 54, 57, 58, 60, 62, 64, 66, 68, and 70,and sequences having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,96%, 97%, 98% or 99% sequence identity thereto, or can be prepared frombiologically active variants such as those described above. The firstprotein segment can include of a full-length polypeptide selected fromthe group consisting of SEQ ID NOS: 2, 4, 6, 8, 10, 12 14, 16, 18, 20,22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 57,58, 60, 62, 64, 66, 68, and 70, sequences having at least 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

Other first protein segments can consist of biologically active portionsof SEQ ID NOS: 2, 4, 6, 8, 10, 12 14, 16, 18, 20, 22, 24, 26, 28, 30,32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 57, 58, 60, 62, 64, 66,68, and 70, sequences having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 96%, 97%, 98% or 99% sequence identity thereto.

The second protein segment can be a full-length protein or a polypeptidefragment. Proteins commonly used in fusion protein construction includeβ-galactosidase, β-glucuronidase, green fluorescent protein (GFP),autofluorescent proteins, including blue fluorescent protein (BFP),glutathione-S-transferase (GST), luciferase, horseradish peroxidase(HRP), and chloramphenicol acetyltransferase (CAT). Additionally,epitope tags can be used in fusion protein constructions, includinghistidine (His) tags, FLAG tags, influenza hemagglutinin (HA) tags, Myctags, VSV-G tags, and thioredoxin (Trx) tags. Other fusion constructionscan include maltose binding protein (MBP), S-tag, Lex a DNA bindingdomain (DBD) fusions, GAL4 DNA binding domain fusions, and herpessimplex virus (HSV) BP16 protein fusions.

These fusions can be made, for example, by covalently linking twoprotein segments or by standard procedures in the art of molecularbiology. Recombinant DNA methods can be used to prepare fusion proteins,for example, by making a DNA construct which comprises a coding regionfor the protein sequence of SEQ ID NOS: 2, 4, 6, 8, 10, 12 14, 16, 18,20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54,57, 58, 60, 62, 64, 66, 68, and 70, sequences having at least 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identitythereto in proper reading frame with a nucleotide encoding the secondprotein segment and expressing the DNA construct in a host cell, as isknown in the art. Many kits for constructing fusion proteins areavailable from companies that supply research labs with tools forexperiments, including, for example, Promega Corporation (Madison,Wis.), Stratagene (La Jolla, Calif.), Clontech (Mountain View, Calif.),Santa Cruz Biotechnology (Santa Cruz, Calif.), MBL InternationalCorporation (MIC; Watertown, Mass.), and Quantum Biotechnologies(Montreal, Canada; 1-888-DNA-KITS).

Pharmaceutical Compositions and Therapeutic Uses

Pharmaceutical compositions of the invention can comprise CD18 signalpeptide (or a portion thereof)—comprising polypeptides of SEQ ID NOS: 2,4, 6, 8, 10, 12 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40,42, 44, 46, 48, 50, 52, 54, 57, 58, 60, 62, 64, 66, 68, and 70, andsequences having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%,97%, 98% or 99% sequence identity thereto polypeptide-based agents ofthe claimed invention in a therapeutically effective amount. The term“therapeutically effective amount” as used herein refers to an amount ofa therapeutic agent to treat, ameliorate, or prevent a desired diseaseor condition, or to exhibit a detectable therapeutic or preventativeeffect. The effect can be detected by, for example, chemical markers orantigen levels. Therapeutic effects also include reduction in physicalsymptoms. The precise effective amount for a subject will depend uponthe subject's size and health, the nature and extent of the condition,and the therapeutics or combination of therapeutics selected foradministration. Thus, it is not useful to specify an exact effectiveamount in advance. However, the effective amount for a given situationis determined by routine experimentation and is within the judgment ofthe clinician. For purposes of the present invention, an effective dosewill generally be from about 0.01 mg/kg to 50 mg/kg or 0.05 mg/kg toabout 10 mg/kg of the SEQ ID NOS: 2, 4, 6, 8, 10, 12 14, 16, 18, 20, 22,24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 57, 58,60, 62, 64, 66, 68, and 70, sequences having at least 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity theretopolypeptide constructs in the individual to which it is administered.

A pharmaceutical composition can also contain a pharmaceuticallyacceptable carrier. The term “pharmaceutically acceptable carrier”refers to a carrier for administration of a therapeutic agent, such asantibodies or a polypeptide, genes, and other therapeutic agents. Theterm refers to any pharmaceutical carrier that does not itself inducethe production of antibodies harmful to the subject receiving thecomposition, and which can be administered without undue toxicity.Suitable carriers can be large, slowly metabolized macromolecules suchas proteins, polysaccharides, polylactic acids, polyglycolic acids,polymeric amino acids, amino acid copolymers, and inactive virusparticles. Such carriers are well known to those of ordinary skill inthe art. Pharmaceutically acceptable carriers in therapeuticcompositions can include liquids such as water, saline, glycerol andethanol. Auxiliary substances, such as wetting or emulsifying agents, pHbuffering substances, and the like, can also be present in suchvehicles. Typically, the therapeutic compositions are prepared asinjectables, either as liquid solutions or suspensions; solid formssuitable for solution in, or suspension in, liquid vehicles prior toinjection can also be prepared. Liposomes are included within thedefinition of a pharmaceutically acceptable carrier. Pharmaceuticallyacceptable salts can also be present in the pharmaceutical composition,e.g., mineral acid salts such as hydrochlorides, hydrobromides,phosphates, sulfates, and the like; and the salts of organic acids suchas acetates, propionates, malonates, benzoates, and the like. A thoroughdiscussion of pharmaceutically acceptable excipients is available inRemington's Pharmaceutical Sciences (Mack Pub. Co., New Jersey, 1991).

Delivery Methods.

Once formulated, the compositions of the invention can be administereddirectly to the subject or delivered ex vivo, to cells derived from thesubject (e.g., as in ex vivo gene therapy). Direct delivery of thecompositions will generally be accomplished by parenteral injection,e.g., subcutaneously, intraperitoneally, intravenously orintramuscularly, myocardial, intratumoral, peritumoral, or to theinterstitial space of a tissue. Other modes of administration includeoral and pulmonary administration, suppositories, implants, andtransdermal applications, needles, and gene guns or hyposprays. Specificoral treatment includes, but is not limited to, the inclusion of thetherapeutic in the animal feed. Dosage treatment can be a single doseschedule or a multiple dose schedule.

Methods for the ex vivo delivery and reimplantation of transformed cellsinto a subject are known in the art and described in e.g., InternationalPublication No. WO 93/14778. Examples of cells useful in ex vivoapplications include, for example, stem cells, particularlyhematopoetic, lymph cells, macrophages, dendritic cells, APCs, or tumorcells. Generally, delivery of nucleic acids for both ex vivo and invitro applications can be accomplished by, for example, dextran-mediatedtransfection, calcium phosphate precipitation, polybrene mediatedtransfection, protoplast fusion, electroporation, encapsulation of thepolynucleotide(s) in liposomes, direct microinjection of the DNA intonuclei, and viral-mediated, such as adenovirus or alphavirus, all wellknown in the art.

In a preferred embodiments, disorders can be amenable to treatment byadministration of a therapeutic agent based on the providedpolynucleotide or corresponding polypeptide. The therapeutic agent canbe administered in conjunction with one or more other agents including,but not limited to, receptor-specific antibodies and/or chemotherapeutic(e.g., anti-neoplastic agents). Administered “in conjunction” includesadministration at the same time, or within 1 day, 12 hours, 6 hours, onehour, or less than one hour, as the other therapeutic agent(s). Thecompositions may be mixed for co-administration, or may be administeredseparately by the same or different routes.

The dose and the means of administration of the inventive pharmaceuticalcompositions are determined based on the specific qualities of thetherapeutic composition, the condition, age, and weight of the patient,the progression of the disease, and other relevant factors. For example,administration of polynucleotide therapeutic compositions agents of theinvention includes local or systemic administration, includinginjection, oral administration, particle gun or catheterizedadministration, and topical administration. The therapeuticpolynucleotide composition can contain an expression constructcomprising a promoter operably linked to a polynucleotide encoding, forexample, SEQ ID NOS: 2, 4, 6, 8, 10, 12 14, 16, 18, 20, 22, 24, 26, 28,30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 57, 58, 60, 62, 64,66, 68, and 70, and sequences having at least 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. Variousmethods can be used to administer the therapeutic composition directlyto a specific site in the body. For example, a target tissue is locatedand the therapeutic composition injected several times in severaldifferent locations within the target tissue. Alternatively, arterieswhich serve a target tissue are identified, and the therapeuticcomposition injected into such an artery, in order to deliver thecomposition directly into the target tissue. X-ray imaging is used toassist in certain of the above delivery methods.

Inventive polypeptide-mediated targeted delivery of therapeutic agentsto specific tissues can also be used. Receptor-mediated DNA deliverytechniques are described in, for example, Findeis et al., TrendsBiotechnol. (1993) 11:202; Chiou et al., Gene Therapeutics: Methods AndApplications Of Direct Gene Transfer (J. A. Wolff, ed.) (1994); Wu etal., J. Biol. Chem. (1988) 263:621; Wu et al., J. Biol. Chem. (1994)269:542; Zenke et al., Proc. Natl. Acad. Sci. (USA) (1990) 87:3655; Wuet al., J. Biol. Chem. (1991) 266:338. Therapeutic compositionscontaining a polynucleotide are administered in a range of about 100 ngto about 200 mg of DNA for local administration in a gene therapyprotocol. Concentration ranges of about 500 ng to about 50 mg, about 1mg to about 2 mg, about 5 mg to about 500 mg, and about 20 mg to about100 mg of DNA can also be used during a gene therapy protocol. Factorssuch as method of action (e.g., for enhancing or inhibiting levels ofthe encoded gene product) and efficacy of transformation and expressionare considerations which will affect the dosage required for ultimateefficacy of the subgenomic polynucleotides. Where greater expression isdesired over a larger area of tissue, larger amounts of subgenomicpolynucleotides or the same amounts readministered in a successiveprotocol of administrations, or several administrations to differentadjacent or close tissue portions of, for example, a tumor site, may berequired to affect a positive therapeutic outcome. In all cases, routineexperimentation in clinical trials will determine specific ranges foroptimal therapeutic effect.

The therapeutic polynucleotides and polypeptides of the presentinvention can be delivered using gene delivery vehicles. The genedelivery vehicle can be of viral or non-viral origin (see generally,Jolly, Cancer Gene Therapy (1994) 1:51; Kimura, Human Gene Therapy(1994) 5:845; Connelly, Human Gene Therapy (1995) 1:185; and Kaplitt,Nature Genetics (1994) 6:148). Expression of such coding sequences canbe induced using endogenous mammalian or heterologous promoters.Expression of the coding sequence can be either constitutive orregulated.

Viral-based vectors for delivery of a desired polynucleotide andexpression in a desired cell are well known in the art. Exemplaryviral-based vehicles include, but are not limited to, recombinantretroviruses (see, e.g., WO 90/07936; WO 94/03622; WO 93/25698; WO93/25234; U.S. Pat. No. 5,219,740; WO 93/11230; WO 93/10218; U.S. Pat.No. 4,777,127; GB Patent No. 2,200,651; EP 0 345 242; and WO 91/02805),alphavirus-based vectors (e.g., Sindbis virus vectors, Semliki forestvirus (ATCC VR-67; ATCC VR-1247), Ross River virus (ATCC VR-373; ATCCVR-1246) and Venezuelan equine encephalitis virus (ATCC VR-923; ATCCVR-1250; ATCC VR 1249; ATCC VR-532), and adeno-associated virus (AAV)vectors (see, e.g., WO 94/12649, WO 93/03769; WO 93/19191; WO 94/28938;WO 95/11984 and WO 95/00655). Administration of DNA linked to killedadenovirus as described in Curiel, Hum. Gene Ther. (1992) 3:147 can alsobe employed.

Non-viral delivery vehicles and methods can also be employed, including,but not limited to, polycationic condensed DNA linked or unlinked tokilled adenovirus alone (see, e.g., Curiel, Hum. Gene Ther. (1992)3:147); ligand-linked DNA (see, e.g., Wu, J. Biol. Chem. 264:16985(1989)); eukaryotic cell delivery vehicles cells (see, e.g., U.S. Pat.No. 5,814,482; WO 95/07994; WO 96/17072; WO 95/30763; and WO 97/42338)and nucleic charge neutralization or fusion with cell membranes. NakedDNA can also be employed. Exemplary naked DNA introduction methods aredescribed in WO 90/11092 and U.S. Pat. No. 5,580,859. Liposomes that canact as gene delivery vehicles are described in U.S. Pat. No. 5,422,120;WO 95/13796; WO 94/23697; WO 91/14445; and EP 0524968. Additionalapproaches are described in Philip, Mol. Cell Biol. 14:2411 (1994), andin Woffendin, Proc. Natl. Acad. Sci. (1994) 91:11581-11585.

Further non-viral delivery suitable for use includes mechanical deliverysystems such as the approach described in Woffendin et al., Proc. Natl.Acad. Sci. USA 91(24):11581 (1994). Moreover, the coding sequence andthe product of expression of such can be delivered through deposition ofphotopolymerized hydrogel materials or use of ionizing radiation (see,e.g., U.S. Pat. No. 5,206,152 and WO 92/11033). Other conventionalmethods for gene delivery that can be used for delivery of the codingsequence include, for example, use of hand-held gene transfer particlegun (see, e.g., U.S. Pat. No. 5,149,655); use of ionizing radiation foractivating transferred gene (see, e.g., U.S. Pat. No. 5,206,152 and WO92/11033).)

Recombinant and Cloned Ruminant Animals:

Inventive CD18-related aspects (e.g. polypeptide-mediated treatment,etc) include producing animals that are naturally resistant to theeffects of Lkt. This is accomplished by, for example, universallyaltering the genotype of an animal, wherein, the native CD18 molecule,which binds to the toxin Lkt, is replaced with the mutant CD18, whichhas limited Lkt binding. Universally altering the genotype of animalincludes cloning of a given animal having the modified genotype.Additionally, the invention encompasses transgenic animals. Transgenicanimals are those that carry a non-native gene that were introduced intothe animal using similar techniques as described herein and those wellknown in the art. Transgenic animals can subsequently be cloned.

Cloned Ruminants:

Cloned fetuses and calves are produced using the chromatin transferprocedure, as described in Kuroiwa et al., (2004) hereby incorporated byreference specifically for its teaching of cloning of cattle. The methodconsists of sequential application of gene targeting by homologousrecombination and rejuvenation of cell lines by production of clonedfetuses. For example, to generate cattle containing a CD18 molecule thathad a Q(−5)G mutation (an exemplary mutation which results in limitedbinding of Lkt to CD18 signal sequence), a male Holstein primary fetalfibroblast line 6594 is transfected with vectors containing the mutatedsignal sequence to replace the native CD18 coding region. This fetalcell line containing the mutated CD18 are established at 40-60 days ofgestation. Certain fetal cell lines that look promising are recloned toproduce calves. To verify that each calf produced contains the mutatedCD18 genotype, applicants collect ear biopsies and establish fibroblastcell lines for genotyping (Richt et al., 2007, hereby incorporated byreference to teach analysis of cells and animals post cloning).Genotyping is done by genomic PCR specific to each gene targeting event,followed by sequence analysis. Additionally, applicants verify the calfphenotype by collecting blood samples and isolating PMNs and ensuringthat anti-signal peptide serum cannot bind to membrane CD18 of PMNs, asdescribed in FIG. 4 and Example 3.

Gene replacement techniques used in the practice of applicant'sinvention includes, but is not limited to, the gene replacementtechniques described in Kuroiwa et al., (2004) hereby incorporated byreference specifically for its teaching of gene replacement techniquesand sequential application of those techniques. For example, thewildtype CD18 molecule is replaced by the exemplary Q(−5)G mutation inthe signal sequence of CD18 that produces a CD18 molecule with acleavable signal sequence. More specifically two different vectors, eachcontaining specific selection cassettes (e.g., cassettes conferringresistance to neomycin or puromycin) are constructed containing themutated CD18 molecule Q(−5)G. The mutated CD18 coding sequence isflanked on both the 5′ and 3′ ends with between 1 and 10 kb of nativesequences, i.e. sequences that flank the CD18 gene in vivo in cattle.These vectors are then transfected into male Holstein primary fetalfibroblast line 6594.

Additionally, using techniques known in the art, practicing thisinvention includes gene therapy. Gene therapy or gene introductionencompasses treating an animal in need thereof with a vector thatcontains a gene sequence encoding the therapeutic to be produced andprovided to the animal via the animal's own protein producingmechanisms. According to certain aspects the invention includesexpressing nucleic acids sequences encoding CD18 polypeptides (e.g., SEQID NOS:2, 4, 6, 8, 10, 12 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34,36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 57, 58, 60, 62, 64, 66, 68, and70, sequences having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,96%, 97%, 98% or 99% sequence identity thereto include glycosylatedforms) by the cell's own machinery.

Antibodies or Antibody Fragments

Agents of the present invention include antibodies and/or antibodyfragments, and in particular aspects, they are specific for (directedagainst): (1) M. haemolytica leukotoxin (Lkt); (2) against Lkt orantigen or portion thereof that is suitable for binding the signalpeptide, and optionally wherein the anti-Lkt antibody can also bind Lktcomplexed with ruminant CD18 signal peptide; (3) or against a CD18signal peptide, or portion thereof comprising from about 13 to about 24contiguous amino acid residues of the first 24 amino acids of theN-terminus of the native (full-length nascent) ruminant CD18 sequence,wherein the polypeptide is suitable to provide for at least one ofbinding to M. haemolytica leukotoxin (Lkt) and abrogation of Lkt-inducedcytolysis; and (4) against signal peptide and able to bind Lkt complexedwith ruminant CD18 signal peptide.

Suitable antibodies may be monoclonal, polyclonal or monoclonalantibodies tailored to a specific ruminant species (in analogy withhumanized antibodies). Antibodies may be derived by conventionalhybridoma based methodology, from antisera isolated from validatedprotein inoculated animals or through recombinant DNA technology.Alternatively, inventive antibodies or antibody fragments may beidentified in vitro by use of one or more of the readily available phagedisplay libraries. Exemplary methods are disclosed herein.

In one exemplary embodiment of the present invention, antibody agentsare monoclonal antibodies that may be produced as follows. Targetproteins in a baculovirus based system. By this method, target proteincDNAs or epitope-bearing fragments thereof are ligated into a suitableplasmid vector that is subsequently used to transfect Sf9 cells tofacilitate protein production. In addition, it may be advantageous toincorporate an epitope tag or other moiety to facilitate affinitypurification of the target protein. Clones of Sf9 cells expressing aparticular protein are identified, e.g., by enzyme-linked immunosorbantassay (ELISA), lysates are prepared and the target protein purified byaffinity chromatography. The purified target protein is, for example,injected intraperitoneally, into BALB/c mice to induce antibodyproduction. It may be advantageous to add an adjuvant, such as Freund'sadjuvant, to increase the resulting immune response.

Serum is tested for the production of specific antibodies, and spleencells from animals having a positive specific antibody titer are usedfor cell fusions with myeloma cells to generate hybridoma clones.Supernatants derived from hybridoma clones are tested for the presenceof monoclonal antibodies having specificity against a particularvalidated protein or fragments thereof. For a general description ofmonoclonal antibody methodology, See, e.g., Harlow and Lane, Antibodies:A Laboratory Manual, Cold Spring Harbor Laboratory (1988).

In addition to the baculovirus expression system, other suitablebacterial or yeast expression systems may be employed for the expressionof a particular target protein or polypeptides thereof. As with thebaculovirus system, it may be advantageous to utilize one of thecommercially available affinity tags to facilitate purification prior toinoculation of the animals. Thus, the target protein cDNA or fragmentthereof may be isolated by, e.g., agarose gel purification and ligatedin frame with a suitable tag protein such as 6-His,glutathione-S-transferase (GST) or other such readily available affinitytag. See, e.g., Molecular Biotechnology: Principles and Applications ofRecombinant DNA, ASM Press pp. 160-161 (ed. Glick, B. R. and Pasternak,J. J. 1998).

In additional embodiments of the present invention, antibody agents areruminantized anti-target protein monoclonal antibodies. The phrase“ruminantized antibody” refers to an antibody derived from anon-ruminant antibody—typically a mouse monoclonal antibody.Alternatively, a ruminantized antibody may be derived from a chimericantibody that retains or substantially retains the antigen-bindingproperties of the parental, non-ruminant, antibody but which exhibitsdiminished immunogenicity as compared to the parental antibody whenadministered to ruminant. The phrase “chimeric antibody,” as usedherein, refers to an antibody containing sequence derived from twodifferent antibodies (see, e.g., U.S. Pat. No. 4,816,567) whichtypically originate from different species. Most typically, chimericantibodies comprise human and murine antibody fragments, generallybovine constant and mouse variable regions.

Because ruminantized antibodies are far less immunogenic in ruminantthan the parental mouse monoclonal antibodies, they can be used for thetreatment of ruminant with far less risk of anaphylaxis. Thus, theseantibodies may be preferred in therapeutic applications that involve invivo administration to a ruminant such as, e.g., use as radiationsensitizers for the treatment of neoplastic disease or use in methods toreduce the side effects of, e.g., cancer therapy.

Ruminantized antibodies may be achieved by a variety of methodsincluding, for example: (1) grafting the non-ruminant complementaritydetermining regions (CDRs) onto a ruminant framework and constant region(a process referred to in the art as “humanizing”), or, alternatively,(2) transplanting the entire non-ruminant variable domains, but“cloaking” them with a ruminant-like surface by replacement of surfaceresidues (a process referred to in the art as “veneering”). In thepresent invention, ruminantized antibodies will include both“ruminantized” and “veneered” antibodies. These methods, in the contextof humanized antibodies, are disclosed in, e.g., Jones et al., Nature(1986) 321:522-525; Morrison et al., Proc. Natl. Acad. Sci., U.S.A.,(1984) 81:6851-6855; Morrison and Oi, Adv. Immunol. (1988) 44:65-92;Verhoeyer et al., Science (1988) 239:1534-1536; Padlan, Molec. Immun.(1991) 28:489-498; Padlan, Molec. Immunol. (1994) 31(3):169-217; andKettleborough, C. A. et al., Protein Eng. (1991) 4:773-83 each of whichis incorporated herein by reference.

The phrase “complementarity determining region” refers to amino acidsequences which together define the binding affinity and specificity ofthe natural Fv region of a native immunoglobulin binding site. See,e.g., Chothia et al., J. Mol. Biol. (1987) 196:901-917; Kabat et al.,U.S. Dept. of Health and Human Services NIH Publication No. 91-3242(1991). The phrase “constant region” refers to the portion of theantibody molecule that confers effector functions. In the presentinvention, mouse constant regions are substituted by human constantregions. The constant regions of the subject humanized antibodies arederived from human immunoglobulins. The heavy chain constant region canbe selected from any of the five isotypes: alpha, delta, epsilon, gammaor mu.

One method of ruminantized antibodies comprises aligning thenon-ruminant heavy and light chain sequences to ruminant heavy and lightchain sequences, selecting and replacing the non-ruminant framework witha ruminant framework based on such alignment, molecular modeling topredict the conformation of the ruminantized sequence and comparing tothe conformation of the parent antibody. This process is followed byrepeated back mutation of residues in the CDR region which disturb thestructure of the CDRs until the predicted conformation of theruminantized sequence model closely approximates the conformation of thenon-ruminant CDRs of the parent non-ruminant antibody. Such ruminantizedantibodies may be further derivatized to facilitate uptake andclearance, e.g., via recpetors in analogy with the use of Ashwellreceptors (see, e.g., U.S. Pat. Nos. 5,530,101 and 5,585,089, bothincorporated herein by reference.

Ruminantized antibodies to a particular target protein can also beproduced using transgenic animals that are engineered to containruminant immunoglobulin loci. In analogy with humanized antibodies forexample, WO 98/24893 discloses transgenic animals having a human Iglocus wherein the animals do not produce functional endogenousimmunoglobulins due to the inactivation of endogenous heavy and lightchain loci. WO 91/10741 also discloses transgenic non-primate mammalianhosts capable of mounting an immune response to an immunogen, whereinthe antibodies have primate constant and/or variable regions, andwherein the endogenous immunoglobulin-encoding loci are substituted orinactivated. WO 96/30498 discloses the use of the Cre/Lox system tomodify the immunoglobulin locus in a mammal, such as to replace all or aportion of the constant or variable region to form a modified antibodymolecule. WO 94/02602 discloses non-human mammalian hosts havinginactivated endogenous Ig loci and functional human Ig loci. U.S. Pat.No. 5,939,598 discloses methods of making transgenic mice in which themice lack endogenous heavy claims, and express an exogenousimmunoglobulin locus comprising one or more xenogeneic constant regions.

Using a transgenic animal described above, an immune response can beproduced to a selected antigenic molecule (e.g., target protein orfragment thereof), and antibody-producing cells can be removed from theanimal and used to produce hybridomas that secrete ruminant monoclonalantibodies. Immunization protocols, adjuvants, and the like are known inthe art, and are used in immunization of, for example, a transgenicmouse as described in WO 96/33735. This publication discloses monoclonalantibodies against a variety of antigenic molecules including IL-6,IL-8, TNFα, human CD4, L-selectin, gp39, and tetanus toxin. Themonoclonal antibodies can be tested for the ability to inhibit orneutralize the biological activity or physiological effect of thecorresponding protein. WO 96/33735 discloses that monoclonal antibodiesagainst IL-8, derived from immune cells of transgenic mice immunizedwith IL-8, blocked IL-8-induced functions of neutrophils. Humanmonoclonal antibodies with specificity for the antigen used to immunizetransgenic animals are also disclosed in WO 96/34096.

For purposes of the present invention, target polypeptides and variantsthereof are used to immunize an animal or transgenic animal as describedabove. Monoclonal antibodies are made using methods known in the art,and the specificity of the antibodies is tested using isolated targetpolypeptides. The suitability of the antibodies for clinical use istested by, for example, exposing HCMV-infected cells to the antibodiesand measuring cell growth and/or phenotypic changes. Ruminant monoclonalantibodies specific for a particular validated protein, or for a variantor fragment thereof can be tested for their ability to inhibit, forexample, cell migration. Such antibodies would be suitable forpre-clinical and clinical trials as pharmaceutical agents for preventingor controlling virus or bacterial-mediated effects, conditions ordiseases.

It will be appreciated that alternative target protein inhibitorantibodies may be readily obtained by other methods commonly known inthe art. One exemplary methodology for identifying antibodies having ahigh specificity for a particular validated protein is the phage displaytechnology.

Phage display libraries for the production of high-affinity antibodiesare described in, for example, Hoogenboom, H. R. et al.,Immunotechnology (1998) 4(1):1-20; Hoogenboom, H. R., Trends Biotechnol.(1997) 15:62-70 and McGuinness, B. et al., Nature Bio. Technol. (1996)14:1149-1154 each of which is incorporated herein by reference. Amongthe advantages of the phage display technology is the ability to isolateantibodies of ruminant origin that cannot otherwise be easily isolatedby conventional hybridoma technology. Furthermore, phage displayantibodies may be isolated in vitro without relying on an animal'simmune system.

Antibody phage display libraries may be accomplished, for example, bythe method of McCafferty et al., Nature (1990) 348:552-554 which isincorporated herein by reference. In short, the coding sequence of theantibody variable region is fused to the amino terminus of a phage minorcoat protein (pIII). Expression of the antibody variable region-pIIIfusion construct results in the antibody's “display” on the phagesurface with the corresponding genetic material encompassed within thephage particle.

A target protein, or fragment thereof suitable for screening a phagelibrary may be obtained by, for example, expression in baculovirus Sf9cells as described, supra. Alternatively, the target protein codingregion may be PCR amplified using primers specific to the desired regionof the validated protein. As discussed above, the target protein may beexpressed in E. coli or yeast as a fusion with one of the commerciallyavailable affinity tags.

The resulting fusion protein may then be adsorbed to a solid matrix,e.g., a tissue culture plate or bead. Phage expressing antibodies havingthe desired anti-target protein binding properties may subsequently beisolated by successive panning, in the case of a solid matrix, or byaffinity adsorption to a validated protein antigen column. Phage havingthe desired target protein inhibitory activities may be reintroducedinto bacteria by infection and propagated by standard methods known tothose skilled in the art. See Hoogenboom, H. R., Trends Biotechnol.,supra for a review of methods for screening for positive antibody-pIIIphage.

Vaccination of Ruminants

Agents of the present invention include compositions that elicit aspecific immune response in a ruminant in need thereof. According tocertain embodiments, the elicitation of the specific immune responsetreats, reduces the likelihood, and/or limits a M. haemolytica infectionand/or the symptoms thereof. In particular aspects, these compositionsthat elicit a specific immune response are: (1) M. haemolyticaleukotoxin (Lkt); (2) Lkt or antigen or portion thereof that is suitablefor binding the signal peptide, and optionally wherein the immuneresponse elicited can also recognize Lkt complexed with ruminant CD18signal peptide; (3) a CD18 signal peptide, or portion thereof comprisingfrom about 13 to about 24 contiguous amino acid residues of the first 24amino acids of the N-terminus of the native (full-length nascent)ruminant CD18 sequence; and/or (4) signal peptide, wherein the elicitedimmune response is capable of binding to Lkt complexed with ruminantCD18 signal peptide.

According to certain embodiments, a composition that elicits a specificimmune response is a vaccine. The terms “vaccine” “vaccination” and“vaccinating” mean the inoculation of a substance or composition (avaccine) into the body of the subject for the purpose of producingimmunity against a disease that is for the purpose of treating orpreventing a disease. Accordingly, vaccination may be therapeutic orprophylactic. By therapeutic vaccination is meant the administration ofa vaccine to a subject already suffering from a M. haemolyticainfection, typically for the purpose of heightening or broadening theimmune response to thereby halt, impede or reverse the progression ofthe disease.

Vaccination in accordance with the invention may provide protectiveimmunity against a M. haemolytica infection to the subject beingvaccinated. That is, the component(s) of the vaccine may elicit aprotective immune response in the subject, for example by inducing theproduction of autoantibodies, innate immunity or adaptive immunityagainst the component(s). As used herein, the term “protective immunity”refers to the ability of a molecule or composition administered to asubject to elicit an appropriate immune response in the subject andthereby provide protection to the subject from the development orprogression of a M. haemolytica infection.

The efficacy of compositions that elicit a specific immune response foruse in accordance with the invention may be enhanced by the use of oneor more adjuvants. Adjuvants capable of enhancing the delivery orprotective or therapeutic efficacy of vaccines (for example by boostingthe immune response produced) are well known to those skilled in theart.

Compositions that elicit a specific immune response may be preparedaccording to methods which are known to those of ordinary skill in theart and accordingly may include a pharmaceutically acceptable carrier,diluent and/or adjuvant. For administration in accordance with thepresent invention, a suitable vaccine may be formulated in apharmaceutically acceptable carrier according to the mode and route ofadministration to be used. The carriers, diluents and adjuvants must be“acceptable” in terms of being compatible with the other ingredients ofthe composition, and not deleterious to the recipient thereof. Typicallya sterile water or isotonic formulation is employed. For example, asuitable isotonic solution is phosphate buffered saline or Ringer'ssolution.

Those skilled in the art will appreciate that the methods andvaccinations contemplated by the present invention may be carried out inconjunction with other therapies or preventative measures for thetreatment or prevention of M. haemolytica infections or symptomsassociated with such diseases. For such combination therapies, eachcomponent of the combination therapy may be administered at the sametime, or sequentially in any order, or at different times, so as toprovide the desired effect. Alternatively, the components may beformulated together in a single dosage unit as a combination product.

Example 1 Materials and Methods

Inhibition of Lkt Binding to, and Cytolysis of Target Cells by thePeptide Analogs of CD18.

Flow cytometric analysis of Lkt binding to target cells, and MTTdye-reduction cytotoxicity assay for detection of Lkt-induced cytolysishave been previously described by Applicants^(4,7). Detection ofinhibition of Lkt binding to, and Lkt-induced cytolysis of, target cellsby peptides in the disclosed studies were performed essentially asdescribed, with the obvious exception that Lkt was pre-incubated withthe peptides before incubation with the target cells.

Peptides.

The nested set of 20-mer peptides spanning aa 1-291 of bovine CD18, andthe N- and C-terminally truncated versions of the Lkt-binding minimalpeptide were synthesized at Sigma-Genosys. An irrelevant peptide(20-mer) derived from major surface protein 1 (MSP1) of Anaplasmamarginale was used as the negative control.

Cloning and Expression of Bovine CD18 Carrying the Mutation Q to G.

The bovine cDNA for CD18²⁸ was previously subcloned into the mammalianexpression vector pCI-neo to yield pMD1⁴. To produce the Q(−5)G mutationin bovine CD18, site-directed mutagenesis was performed using theGeneTailor™ site-directed mutagenesis system (Invitrogen). CD18 sequenceafter the point mutation was checked by DNA sequencing. Transfection ofP815 cells with Lipofectamine™ 2000 was carried out according to thesupplier's recommendations.

Cloning and Expression of Bovine CD18 Carrying the ‘FLAG’ Epitope at theCleavage site.

The GeneTailor™ site-directed mutagenesis system (Invitrogen) was usedto insert the ‘FLAG’ epitope (DYKDDDDK) into the vector pMD1 carryingbovine CD18 cDNA, at the signal peptide cleavage site (between aa 22 and23). The insertion was carried out in two steps (12 bp at a time)according to the manufacturer's instructions. The insertion of ‘FLAG’epitope into CD18 was confirmed by DNA sequencing. The vector carryingthe ‘FLAG’-tagged CD18 was transfected into P815 cells withLipofectamine™ 2000 according to the manufacturer's protocol.

Statistical Analysis.

One-way ANOVA was employed to determine whether the differences in %inhibition caused by the different peptides are statisticallysignificant.

Preparation of Lkt.

Production of Lkt from M. haemolytica A1 has been previously describedby Applicants²⁹. The undiluted toxin preparation contained 640 Units oftoxin per ml. All experiments were performed with the same batch oftoxin aliquoted and frozen at −20° C.

Cell Lines and Antibodies.

The cell lines P815 (murine mastocytoma), and BL3 (bovine lymphoma3),were propagated in complete Dulbecco's minimum Eagle's medium or RPMI1640, respectively, supplemented with 10% fetal bovine serum, 2 mML-glutamine, and 20 ug/ml of gentamicin (complete medium). Thetransfectant 2B2, expressing full-length bovine CD18 on the cellsurface, was previously developed in Applicants' laboratory bytransfecting P815 with cDNA for bovine CD18⁴. The transfectants wereselected and propagated in the complete DMEM together with 500 ug/ml ofGeneticin (Invitrogen). PMNs were isolated from peripheral blood bydensity gradient centrifugation using Ficoll-Paque (Amersham PharmaciaBiotech.), followed by hypotonic lysis of the erythrocyte pellet, aspreviously described³⁰. Anti-bovine CD18 monoclonal antibody (mAb)BAQ30A was obtained from Washington State University Monoclonal AntibodyCenter. The Lkt-non-neutralizing mAb MM605 (IgG2a) was previouslydeveloped in Applicants' laboratory²⁹. FITC-conjugated MM-605 was usedin flow cytometry to detect Lkt-binding⁷.

Peptides.

A nested set of 20-mer peptides spanning aa 1-291 of bovine CD18 wassynthesized with either 6 or 15 aa overlap. Once the peptide whichinhibits Lkt-induced cytotoxicity was identified, another set ofpeptides were synthesized with N-terminal truncation by dropping one aaat a time while keeping the C-terminal aa constant. Once the N-terminalaa of the minimal peptide was identified, another set of peptides weresynthesized with C-terminal truncation by dropping one aa at a timewhile keeping the N-terminal aa constant. Peptides were purchased fromSigma Genosys. All the peptides were referred to by the sequence numberof their first amino acid. An irrelevant peptide (20-mer) derived frommajor surface protein 1 (MSP1) of Anaplasma marginate was used as thenegative control. All the peptides were resuspended in dimethysulfoxide(ATCC) at a concentration of 1 mg/ml, aliquoted and stored at −20° C.

Detection of Inhibition of Lkt-Induced Cytolysis of Target Cells by thePeptide Analogs of CD18.

The MTT [3-(4,5-dimethylthiazoyl-2-YI]-2,5-diphenyl tetrazolium bromide;Sigma] dye reduction cytotoxicity assay for detection of Lkt-inducedcytolysis of target cells has been previously described by Applicants³⁰.This assay measures the ability of the endoplasmic reticulum-residentenzymes in viable cells to convert a tetrazolium dye into a purpleformazan precipitate, which is later dissolved in acid isopropanol. Theoptical density (OD) of the end product, representing the intensity ofthe purple color which developed, is directly proportional to theviability of the cells. The percent cytotoxicity was calculated asfollows:

% cytotoxicity=[1−(OD of toxin-treated cells/OD of toxin-untreatedcells)]×100.

Inhibition of Lkt-induced cytolysis of target cells by the peptideanalogs was detected by the MTT assay with the obvious exception thatLkt was pre-incubated with the peptides before incubation with thetarget cells. Lkt was used at the dilution that causes 50% cytolysis oftarget cells. The percent inhibition of cytolysis was calculated asfollows:

% Inhibition of cytolysis=[(1−% cytolysis in the presence of peptide)/%cytolysis in the absence of peptide]×100.

Flow Cytometric Analysis of Inhibition of Lkt Binding to Target Cells.

Flow cytometric analysis of Lkt binding to target cells has beenpreviously described by Applicants. Inhibition of Lkt binding to targetcells by the peptides in the presently disclosed studies was performedessentially as previously described by Applicants with the obviousexception that Lkt was pre-incubated with the peptides before incubationwith the target cells.

Flow Cytometric Analysis of the Cell Surface Expression of CD18 onTransfectant.

Transfectants were examined for the cell surface expression of CD18using anti-CD18 MAb by flow cytometry, as previously described⁴.

Example 2 Peptide Analogs of Bovine CD18 Inhibited Lkt Binding to, andCytolysis of, Ruminant PMNs

Applicants previously mapped the Lkt binding site on bovine CD18 to liebetween amino acids (aa)1-291⁷. In this EXAMPLE, inhibition ofLkt-induced cytolysis of target cells by a nested set of 20-mer peptidesspanning aa 1-291 of bovine CD18 was used to determine the Lkt bindingsite on bovine CD18. Lkt-induced cytolysis of bovine PMNs was stronglyinhibited by two peptides, P1 and P5, containing aa 1-20 and 5-24,respectively, at a concentration of 5 ug/50 ul (FIG. 1 a).

Specifically FIG. 1 a shows, according to particular exemplary aspects,that the CD18 signal peptide analog P5 (aa 5-24) inhibits Lkt-inducedcytolysis of bovine PMNs. Inhibition of Lkt-induced cytolysis of bovinePMNs by a nested set of peptides (20-mers) spanning the aa 1-291 wastested by the MTT dye-reduction cytotoxicity assay. All data areexpressed as mean±s.d. (n=3). a. Inhibition of cytolysis of bovine PMNsby Lkt. The peptides are designated by the sequence number of theirfirst amino acid. Since the most pronounced aa sequence difference inthe CD18 of ruminants and non-ruminants was observed in the N-terminalregion, the point of origin of first 5 peptides was staggered by 5 aa,and the rest of the peptides were staggered by 14 aa. A 20-mer peptidederived from major surface protein 1 of Anaplasma marginale (MSP-1) wasused as the negative control. The peptides were used at a concentrationof 5 μg/50 μl. % Inhibition was calculated as described in the Methods.b. Determination of the concentration of P1 and P5 that gives 50%inhibition of Lkt-induced cytolysis of bovine PMNs. The peptides P1 andP5 were used in the inhibition assay at concentrations ranging from 3.1ug/ml to 100 ug/ml. c. Specificity of inhibition of Lkt-inducedcytolysis of bovine PMNs by the peptide P5. Two peptides containing thesame aa as the peptide P5, but in a randomly scrambled sequence, wereused in the cytotoxicity assay along with P5.

Comparison of the concentration of peptides P1 and P5 which causes 50%inhibition of Lkt-induced cytolysis of bovine PMNs revealed the potencyof peptide P5 to be higher than that of peptide P1 (12 ug/ml versus 26ug/ml; FIG. 1 b). Similar results were obtained with PMNs of otherruminants (goats, domestic sheep, wild sheep, and deer) as well (datanot shown). Two other peptides containing the same as P5, but in arandomly scrambled sequence, failed to inhibit Lkt-induced cytolysis oftarget cells indicating that the inhibition of Lkt-induced cytolysis ofbovine PMNs by the peptide P5 is specific (FIG. 1 c).

Flow cytometric analysis confirmed the inhibition of Lkt binding tobovine PMNs by peptide P5 (FIG. 2).

Specifically, FIG. 2 shows, according to particular exemplary aspects,that peptide P5 inhibits Lkt binding to bovine PMNs. Binding of Lkt tobovine PMNs was tested by flow cytometry, in the absence of any peptide(a & b; black histogram), or in the presence of peptide P5 (a; grayhistogram), or an irrelevant peptide (b; gray histogram) by flowcytometry. Shaded histogram represents the background fluorescence givenby the cells. The x and y axis show the fluorescence intensity and thenumber of cells, respectively. Results of one representative experimentout of three are shown.

Inhibition of Lkt-induced cytolysis of target cells by shorter versionsof peptides derived from P5 by N- and C-terminal truncations identifiedaa 5-17 of ruminant CD18 as the sequence that serves as the receptor forLkt. Hence the peptide made up of aa 5-17 is the minimal peptide analogof ruminant CD18 that effectively inhibits Lkt-induced cytolysis ofruminant PMNs and other leukocyte subsets (FIG. 3).

Specifically, FIG. 3 shows, according to particular exemplary aspects,that N- and C-terminal truncations of peptide P5 identifies the minimalpeptide sequence of bovine CD18 bound by Lkt as aa 5-17. Inhibition ofLkt-induced cytolysis of BL-3 cells by N-terminally truncated orC-terminally truncated versions of peptide P5 were determined by the MTTdye-reduction cytotoxicity assay. Peptides were used at concentrationsranging from 1.56 to 100 um/ml. All data are expressed as mean±s.d.(n=3). a. Inhibition of Lkt-induced cytolysis of BL-3 cells by peptideswith N-terminal truncation. The peptides used were: P5 (aa 5-24); P6 (aa6-24); P7 (aa 7-24); P8 (aa 8-24); P9 (aa 9-24); P10 (aa 10-24); P11 (aa11-24); P12 (aa 12-24); P6 (aa 6-24); P6 (aa 6-24). b. Inhibition ofLkt-induced cytolysis of BL-3 cells by peptides with C-terminaltruncation. Results of the inhibition assay in a above, identified theN-terminal aa of the minimal peptide sequence as aa #5. Therefore, theC-terminally truncated versions of peptide P5 were synthesized with aa#5 as the N-terminal aa. The peptides used were: P24 (aa 5-24); P23 (aa5-23); P22 (aa 5-22); P21 (aa 5-21); P20 (aa 5-20); P19 (aa 5-19); P18(aa 5-18); P17 (aa 5-17); P16 (aa 5-16). Results of the inhibition assayin a and b above, identified the minimal peptide sequence of bovine CD18bound by leukotoxin as aa 5-17. c. Comparison of inhibition given by thepeptide P5 (aa 5-24) and the minimal peptide P17 (aa 5-17).

Example 3 The Amino Acid Sequence of the Bovine CD18 Peptide Analog thatInhibits Lkt-Induced Cytolysis was Shown to be from the Signal Sequenceof CD18

The amino acids 5-17 constitute the bulk of the predicted signalsequence (amino acids 1-22) of CD18. As appreciated in the relevant art,paradigm dictates that signal peptides of plasma membrane proteins arecleaved by the signal peptidase in the ER¹⁻³. Therefore Applicantschecked for the presence of the signal peptide on mature cell surfaceCD18.

As shown in FIG. 4, flow cytometric analysis with an anti-serum specificfor the signal peptide of bovine CD18 indicated that the CD18 moleculesare on the surface of ruminant PMNs and, indeed, have the signal peptideintact.

Specifically, FIG. 4 shows, according to particular exemplary aspects,that anti-signal peptide serum binds to membrane CD18 of PMNs of allruminants tested. PMNs from the indicated animals were tested by flowcytometry for the binding of a chicken anti-serum (1/1000 dilution)developed against a synthetic peptide spanning aa 5-19 of bovine CD18signal peptide. Serum from un-immunized chicken, and MDBK cells, wereused as the negative controls for the serum and cells, respectively. Thedifferent panels show the results of flow cytometry with PMNs of (a)cattle; (b) goats; (c) domestic sheep; (d) wild sheep; (e) deer; (f)Jurkat cells (human T-lymphoma); (g) RAW264.A cells (mouse macrophage);(h) HEK-293 (human embryonic kidney). Shaded histogram: cells only;silver histogram: cells treated with serum from un-immunized chicken;gray histogram: cells treated with anti-serum against signal peptide ofbovine CD18. Results of one representative experiment out of three areshown.

Example 4 The Signal Peptide of Ruminant CD18 was Shown not to beCleaved from the Mature Protein

To confirm the fact that the signal peptide of bovine CD18 is notcleaved, Applicants introduced a minigene encoding the ‘FLAG’ epitope²¹at the putative signal peptide cleavage site (between aa 22 and 23).Transfectants stably expressing bovine CD18 containing the ‘FLAG’epitope between aa 22 and 23 were tested with two monoclonal antibodies(MAbs), M1 and M2, specific for the ‘FLAG’ epitope. M1 recognizes thefree N-terminal end of ‘FLAG’, while M2 recognizes ‘FLAG’ irrespectiveof its sequence context²¹.

As shown in FIG. 5, the MAb M2, but not M1, bound to the transfectantsexpressing ‘FLAG’-containing CD18 confirming that the signal peptide ofbovine CD18 is indeed not cleaved.

Specifically, FIG. 5 shows, according to particular exemplary aspects,that the signal peptide of bovine CD18 is not cleaved. a. Introductionof the ‘FLAG’ epitope at the signal peptide cleavage site and itsdetection by two monoclonal antibodies. The ‘FLAG’ epitope (DYKDDDDK)introduced at the signal peptide cleavage site (between aa 22 and 23)can be detected by two anti-‘FLAG’ epitope antibodies M1 and M2. M1recognizes the free N-terminal end of ‘FLAG’ (if cleavage occurs), whileM2 recognizes ‘FLAG’ irrespective of its sequence context. b. Flowcytometric analysis of bovine CD18 expression. The untransfected parentcells P815 (shaded histogram), or P815 cells transfected with eitherbovine CD18 (2B2; black histogram) or bovine CD18 containing the ‘FLAG’epitope at the cleavage site (BFL; gray histogram), were tested forexpression of bovine CD18 by flow cytometric analysis with ananti-bovine CD18 monoclonal antibody. C.-d. Flow cytometric analysis ofexpression of the ‘FLAG’ epitope. The transfectants 2B2 and BFL weretested for the expression of the ‘FLAG’ epitope with the monoclonalantibody M2 (c.) and M1 (d.). Results of one representative experimentout of three are shown.

Example 5 The Signal Peptide of CD18 of Non-Ruminants was Shown toContain ‘Cleavage-Conducive’ Glycine, Whereas that of Ruminants wasShown to Contain ‘Cleavage-Inhibiting’ Glutamine at Position −5 Relativeto the Cleavage Site

Applicants' finding that a sequence (aa 5-17) within the signal peptideof ruminant CD18 serve as the receptor for M. haemolytica Lkt, and thefact that the cytolytic activity of Lkt is absolutely specific forruminant leukocytes, prompted examination of the signal peptide of CD18of ruminants and non-ruminants. The amino acid sequences of CD18 ofeight ruminants and five non-ruminants were compared (FIG. 6).

Specifically, FIG. 6 shows, according to particular exemplary aspects,that the signal peptide of CD18 of ruminants contain glutamine at aaposition −5 relative to the cleavage site, whereas that of non-ruminantscontain glycine. a. The “−3,−1, rule” for cleavage of signal peptides(Von Hejne²²). b. Comparison of signal peptide sequences of ruminantsand non-ruminants (GenBank Accession #: cattle: M81233; bison: EU553919;buffalo: AY842449; goat: AY452481; domestic sheep: DQ470837; wild sheep:DQ104444; deer: EU623794; elk: EU553918; human: NM0002211; chimpanzee:NM001034122; mouse: X14951; rat: NM001037780; pig: U13941). DS, BHS andchimp denote domestic sheep, wild sheep (bighorn sheep) and chimpanzee,respectively. Arrow indicates the signal peptide cleavage site.

The predicted signal sequence of both the ruminant and non-ruminant CD18contains 22 aa. The “−3-1 rule” of Von Hejne²² for signal peptidecleavage calls for the presence of amino acids with small unchargedamino acids at position −1 and −3 relative to the cleavage site. Bothruminant and non-ruminant CD18 signal peptides conform to this rule. Theamino acid residue at position −5 could also determine whether thesignal peptide gets cleaved or not²³. Helix-breaking residues glycineand proline are conducive for signal peptide) cleavage²³. Arginine isalso conducive to signal peptide cleavage^(22,23). Glutamine on theother hand has been shown to inhibit cleavage of signal peptide²³.Astonishingly, CD18 of all five non-ruminants examined contained the‘cleavage-conducive’ glycine (humans, mice, rats, and chimpanzees) orarginine (pigs), while CD18 of all eight ruminants examined contained‘cleavage-inhibiting’ glutamine.

Example 6 Transfectants Expressing Bovine CD18 Containing the Mutationof Glutamine at −5 Position to Glycine were Shown to be not Susceptibleto M. haemolytica Lkt-Induced Cytolysis

The observation that the signal peptide of CD18 of ruminants(Lkt-susceptible) contains Q at −5 position whereas that ofnon-ruminants (Lkt-non-susceptible) contains G raised the question as towhether site-directed mutagenesis of Q to G [Q(−5)G] would result in theabrogation of Lkt-induced cytolysis of transfectants expressing Q(−5)Gmutation in the signal peptide of CD18. Indeed, as disclosed herein,that is precisely what was found. The Q(−5)G mutation in the signalpeptide of bovine CD18 abrogated Lkt-induced cytolysis of thetransfectants expressing the mutated CD18 (FIG. 7).

Specifically, FIG. 7 shows, according to particular exemplary aspects,that mutation of glutamine at position −5 of the signal peptide ofbovine CD18 to glycine abrogates Lkt-induced cytolysis of transfectantsexpressing CD18 with Q(−5)G mutation. Transfectants expressing bovineCD18 (2B2), those expressing bovine CD18 containing the Q(−5)G mutation(BQG), and the parent cells (P815) were tested for susceptibility toLkt-induced cytolysis by the MTT dye-reduction cytotoxicity assay. Alldata are expressed as mean±s.d. (n=3).

In summary, particular aspects disclosed herein demonstrate for thefirst time that the aa 5-17 within the signal peptide of ruminant CD18serve as the receptor for M. haemolytica Lkt, and that the failure ofthe signal peptide to be cleaved from mature CD18 molecules renders theruminant leukocytes susceptible to Lkt.

Dileepan et al.^(24,25) has previously reported that Lkt binding sitelies within aa 500-600, more precisely between aa 541-581 of bovineCD18. The present results indicate that this conclusion is erroneous forvarious reasons.

First, two different sets of synthetic peptides spanning aa 500-600failed to inhibit Lkt-induced cytolysis of bovine PMNs (FIG. 8).Specifically, FIG. 8 shows, according to particular exemplary aspects,that peptides spanning aa 500-600 of bovine CD18 fail to inhibitLkt-induced cytolysis of bovine PMNs. a. Nested sets of peptidesspanning aa 500-600. One set (#1) of peptides (20-mer) spanning aa500-600 were synthesized and tested in the inhibition assay. Since noneof the peptides inhibited Lkt-induced cytolysis of bovine PMNs, anotherset (#2) of peptides with points of origin different from that of thefirst set were synthesized. b & c. Inhibition of Lkt-induced cytolysisof bovine PMNs by the nested set of peptides was tested by the MTTdye-reduction cytotoxicity assay. All data are expressed as mean±s.d.(n=3).

Second, synthetic peptides containing the signal sequence aa 5-17completely inhibited Lkt-induced cytolysis of PMNs of bovine (FIG. 1)and other ruminants. If Lkt bound to CD18 between aa 541 and 581, onewould expect to see cytolysis of target cells when Lkt is incubated witha synthetic peptide representing the signal sequence (aa 5-17).

Third, Applicants; transfectants expressing CD18 containing the Q(−5)Gmutation in the signal peptide are not lysed by Lkt although the aa 500to 600 are intact in the CD18. According to particular aspects, thefailure of Dileepan et al^(24,25) to identify aa 5-17 in the signalpeptide as the Lkt binding region was likely due to the fact that theirtransductants were developed with K562 cells. According to furtheraspects, K562 cells transfected with bovine CD18 do not express CD18with intact signal peptide (Applicants' unpublished observations).Therefore, according to additional aspects, K562 cells likely carry asignal peptidase that cleaves the signal peptide in spite of thepresence of Q at position −5. K562 is a poorly characterized cell-line,and conflicting reports regarding the lineage of this cell-line can befound in the literature^(26,27). Applicants' studies indicate that thefindings of Dileepan et al.^(24,25) are unique to bovine CD18transductants developed with K562 cells, and do not reflect themolecular events occurring in ruminant leukocytes.

According to particular aspects, the replacement of‘cleavage-inhibiting’ Q at −5 position with ‘cleavage-conducive’ Glikely results in the cleavage of the signal peptide and the resultantloss of susceptibility of the transfectants to Lkt-induced cytolysis.According to additional aspects, abrogation of cytolysis is not due tocleavage of signal peptide, but rather due to conformational changescaused by the replacement of Q with G. According to further aspects,both effects are involved. Irrespective of the molecular basis ormechanism underlying the abrogation of cytolysis, the exemplary Q(−5)Gmutation embodiment provides for a hitherto unavailable technology to,among other things, clone cattle and other ruminants expressing CD18without the signal peptide on their leukocytes, and hence provideanimals less susceptible to pneumonic pasteurellosis, and which willsave millions of dollars annually with world-wide benefit.

Example 7 Endobronchial Inoculation of a Peptide Analog of CD18 was anEffective Inhibitor of M. haemolytica-Caused Pneumonia in a CalfChallenge Model Overview.

In this Example, a study was conducted that confirmed the ability of thepeptide spanning amino acids 5-17 of bovine CD18 to inhibit or mitigatethe disease caused by M. haemolytica, in a calf challenge model.

Leukotoxin (Lkt) produced by Mannheimia haemolytica is the majorvirulence factor of this organism. Lkt-induced cytolysis anddegranulation of alveolar macrophages and polymorphonuclear leukocytesis responsible for the acute inflammation and lung injury characteristicof pneumonia caused by M. haemolytica. Applicants identified a peptideanalog of CD18 (P17, spanning amino acids 5-17) that effectivelyinhibited Lkt-induced cytolysis of ruminant leukocytes in in vitrocytotoxicity assays (Shanthalingam and Srikumaran, 2009). The objectiveof this study was to determine the ability of this peptide to inhibit ormitigate lung lesions in a calf challenge model of M. haemolytica. Threegroups of four calves each were endobronchially inoculated withlogarithmic phase cultures of M. haemolytica (5×10⁹ CFU per 10 ml ofculture medium) alone (Group I), or along with a control peptide (GroupII), or with the CD18 peptide analog P17 (Group III). Animals wereobserved for clinical signs at different time points, euthanized at 90hours post-inoculation, and necropsied. The total clinical diseasescores for Group III calves were lower than those for group I and II atall time points except 48 hours. This difference was statisticallysignificant (P<0.05) at 24 hours post-inoculation. All the calvespresented gross pulmonary lesions consistent with fibrinonecroticpneumonia characteristic of M. haemolytica infection. The difference inpercent volume of lungs exhibiting gross pneumonic lesions among thethree groups was not statistically significant (P=0.9). However, M.haemolytica isolated from the lungs of Group III calves was 100- to1000-fold less than those isolated from the calves in Group I and GroupII. This difference, expressed as CFU of M. haemolytica per g of lungtissue, was statistically significant (P<0.001) indicating that the CD18peptide analog reduced leukotoxic activity in the lungs enabling moreeffective bacterial clearance by the phagocytes.

In particular aspects, prolonging the presence and activity of the CD18peptide analog in the lungs using a nanoparticle delivery system such ascrystallized dextran microspheres enhances its protective ability.

Materials and Methods.

Preparation of M. haemolytica Inoculum for Endobronchial Challenge.

M. haemolytica serotype-1 strain SH789, isolated from the pneumonic lungof a calf, was streaked on blood agar plate and incubated overnight at37° C. The following day few colonies were transferred to 3 ml ofpre-warmed brain heart infusion (BHI) broth and incubated for 3 hours at37° C. with constant shaking (200 cycles/minute). Two BHI agar plateswere ‘lawned’ with this bacterial culture using sterile cotton swabs andincubated overnight at 37° C. The following day (day of inoculation) M.haemolytica was harvested from the BHI agar plates and transferred to 40ml of pre-warmed BHI broth in a 250 ml flask, and incubated for 2.5hours at 37° C. with constant shaking to obtain cultures in thelogarithmic phase of growth. The culture was centrifuged at 6000×g at20° C. for 30 minutes and the pellet was washed once with RPMI 1640(without phenol red) medium. The bacterial pellet was re-suspended in 4ml of RPMI 1640 (without phenol red), and 1 ml of this culture was addedto 50 ml of pre-warmed RPMI 1640 (without phenol red) containingL-glutamine (1 ml L-glutamine/100 ml RPMI) in a 250 ml flask. Bacteriawere incubated for 3 hours at 37° C. with constant shaking to obtainlogarithmic phase culture and the optical density (OD) was measured. Theculture was appropriately diluted to obtain a concentration of 1×10⁹ CFUof M. haemolytica per ml. Five ml of this preparation per calf was usedfor endobronchial challenge. The bacterial concentration was confirmedthe following day by culturing diluted aliquots of the inoculum on BHIagar and counting the resulting colonies.

Peptides.

The peptide (P17) containing amino acids 5-17 of bovine CD18(NH₂-RPQLLLLAGLLAL-OH) (SEQ ID NO.:71), and the peptide (PSC) containingthe same amino acids as peptide P17 but in a randomly scrambled sequence(NH₂-LRALLPLQLLAGL-OH) (SEQ ID NO.:72), were synthesized at Neopeptide(Cambridge, Mass.). Both peptides were re-suspended in dimethysulfoxide(ATCC) at a concentration of 20 mg/ml and stored at −20° C. until used.Based on the results of in vitro neutralization of Lkt by peptide P17,each calf was endobronchially inoculated with 2 mg of peptide in 5 ml ofRPMI mixed with 5×10⁹ CFU of M. haemolytica in 5 ml of RPMI.

In Vitro Neutralization of Lkt.

Five ml aliquots of M. haemolytica containing 1×10⁹ CFU/ml of RPMI 1640were mixed with 5 ml aliquots of the peptide (P17 or PSC) at aconcentration of 5, 4, or 2 mg per 5 ml of RPMI 1640 (without phenolred), and incubated for 4-5 hours at 37° C. with constant shaking. Thebacteria were removed from the culture by centrifugation (13, 500×g for20 min at 4° C.), and the supernatant fluid was filter-sterilized andstored at −20° C. until tested by the cytotoxicity assay for leukotoxicactivity.

Detection of Lkt-Induced Cytolysis of Target Cells.

The MTT [3-(4,5-dimethylthiazoyl-2-YI)-2,5-diphenyl tetrazolium bromide;Sigma] dye reduction cytotoxicity assay for detection of Lkt-inducedcytolysis of target cells has been previously described by us (Gentryand Srikumaran, 1991). This assay measures the ability of theER-resident enzymes in viable cells to convert a tetrazolium dye into apurple formazan precipitate, which is later dissolved in acidisopropanol. The optical density (OD) of the end product, representingthe intensity of the purple color developed, is directly proportional tothe viability of the cells. Briefly, the target cells were re-suspendedin colorless RPMI 1640 (without phenol red) at a concentration of 5×10⁶cells ml⁻¹, and seeded into 96 well round bottom microtiter plates (50ul/well) containing the serially diluted Lkt in duplicates and incubatedat 37° C. for 1 hour. Cells were centrifuged at 600×g for 5 minfollowing incubation, and the supernatant fluid was discarded. The cellpellets were re-suspended in 100 ul of colorless RPMI 1640 and 20 ul of0.5% MTT dye were added to each well. After 1 hour of incubation at 37°C., the plates were centrifuged at 600×g for 5 min and the supernatantfluid was removed. The formazan precipitate was thoroughly dissolved in100 ul acid isopropanol and the OD of the samples was measured using anELISA reader at 540 nm. The percent cytotoxicity was calculated asfollows: % cytotoxicity=[1−(OD of toxin-treated cells/OD oftoxin-untreated cells)]×100.

Detection of Inhibition of Lkt-Induced Cytolysis of Target Cells by theLkt-Neutralizing Abs in Serum.

For Lkt neutralization, 50 ul of toxin preparation at a 50% toxicity endpoint titer of 40 Units/ml was incubated with 50 ul of serum (2 folddilutions starting at 1:20) at 4° C. for 1 hour. Bovine lymphoma cells(BL3; 5×10⁶/ml) were added, and the MTT assay was performed as describedabove. The percent inhibition of cytolysis was calculated as follows: %Inhibition of cytolysis=[1−(% cytolysis in the presence of serum/%cytolysis in the absence of serum)]×100.

Animal Inoculation.

All experimental protocols were approved by the Institutional AnimalCare and Use Committee (IACUC) at Washington State University before theonset of the study. Twelve Holstein calves were randomly assigned tothree experimental groups. Calves were matched for age when assignedinto three groups. Prior to inoculation, serum samples and pharyngealand nasal swabs were collected from all the calves. Group I calvesreceived endobronchial injections of 5×10⁹ CFU of M. haemolytica in 10ml of RPMI. Group II calves received 5×10⁹ CFU of M. haemolytica and 2mg of peptide PSC in 10 ml of RPMI. Group III calves received 5×10⁹ CFUof M. haemolytica and 2 mg of peptide P17 in 10 ml of RPMI. The inoculumwas flushed down with an additional 10 ml of RPMI in all calves.Clinical disease in each calf was scored at different time pointspost-inoculation. Calves were humanely euthanized 90 hourspost-inoculation, and the percent volume of lungs exhibiting grosspulmonary pathology was calculated using morphometric methods.

Scoring of Clinical Disease.

Physical examination of each calf was performed immediately prior toexperimental infection and at 6, 18, 24, 42, 48, 66, 72, and 90 hourspost-inoculation. Signs of clinical disease were allocated pointsaccording to the scoring system followed by Malazdrewich et al (2003;Table 3).

TABLE 3 Evaluation and scoring of clinical signs Clinical signs Clinicalscore Body temperature >103.5° F. 2 Inappetance 1 Lethargy/depression 1Marked weakness/recumbency 2 Moribund state 3 Cough 1 Nasal discharge 1Respiratory rate >60 breaths/min 1 Dyspnea 2 Abnormal breath sounds onauscultation 1

Serotyping.

M. haemolytica isolated from pharyngeal and nasal swabs prior toinoculation and from lung tissue at necropsy, were typed usinganti-serotype A1 serum (kindly provided by Dr. Robert Briggs, NationalAnimal Disease Center, Ames, Iowa). One milliliter of fresh culture wascentrifuged at 6800×g for 3 minutes and the pellet was re-suspended in100 μl of Hanks' balanced salt solution (HBSS) medium containing 0.25%of bovine serum albumin (BSA). Twenty-five μl of culture was then placedon the agglutination plate. Anti-A1 specific serum was diluted in HBSSmedium containing 0.25% of BSA and 25 μl of the diluted serum at 1/16dilution was added to the bacteria and mixed by gentle rocking of theplate. The reference A1 strain and culture media were used as thepositive and negative controls, respectively.

Quantitation of Gross Pulmonary Pathology.

On necropsy lungs were removed and gross pneumonic lesion development ineach lobe of lung was observed. The entire lung from each animal wassliced at 1 cm thickness and the total and pneumonic lesion areas weretraced onto transparent acetate sheets. The traced portions were scannedinto ImgaeJ, NIH Image (National Institute of Health; Nethesda, Md.),which was used to measure areas representing both the total serialsection and the gross pneumonic lesions within it. Measured areas foreach serial section were used to calculate the volume of each lung andthe grossly pneumonic regions within it using Simpson's rule: V=(⅓) h[(A₀+A_(n))+4(A₁+A₃+ . . . +A_(n-1))+2 (A₂+A₄+ . . . +A_(n-2))] where Vis the total or pneumonic lung volume, h is the thickness of each slicein centimeters, and A, A₀, A₁, A₂, A_(n) represent measured lung orpneumonic lesion areas for lung slices 0, 1, 2, n. These values werethen used to calculate the percent volume of the lung exhibiting grosspulmonary pathology in each calf.

Lung tissues from representative gross lesions in each calf werecollected for histopathological evaluation. Tissue samples were fixed in10% neutral buffered formalin and embedded in paraffin using standardtechniques. After routine processing, 5 μm tissue sections were stainedwith hematoxylin and eosin and used for subjective, non-quantitativehistopathological examination.

Re-Isolation of M. haemolytica from Pneumonic Lungs.

Using aseptic techniques, fresh lung samples and pharyngeal and nasalswabs were obtained for isolation and characterization of bacteriaincluding M. haemolytica and other Pasteurella species. Serotyping of M.haemolytica isolates were performed using anti-serotype A1 specificsera.

Bacteriological Examination.

Small samples of tissue (1 g) were obtained from affected regions fromthe same lobe. The samples were homogenized into 3 mls of RPMI 1640 anddiluted 10-fold (1×10⁻¹ to 1×10⁻⁶). Ten μl aliquots of each dilutionwere applied to BHI agar plates on 5 spots and incubated at 37° C.overnight and viable counts were determined. Representative colonieswere checked for M. haemolytica by colony PCR. Primers used were asfollows: forward 5′-AGAGGCCAATCTGCAAACCTC-3′ (SEQ ID NO.:73) and reverse5′-GTTCGTATTGCCCAACGCCG-3′ (SEQ ID NO.:74). Counts were expressed as CFUof M. haemolytica/g of lung tissue.

Statistical Analysis.

Clinical scores and the percent volume of the lung exhibiting grosspneumonic lesions were expressed as the mean±SEM. Clinical scoresbetween the groups were compared using one-way analysis of variancetests (ANOVA). Pneumonic lung scores and the quantity of M.haemolytica/g of lung tissue of all three groups were also comparedusing one-way ANOVA. Differences were considered significant at a valueof P<0.05.

Results

Co-Incubation of Peptide P17 with In Vitro Cultures of M. haemolyticaAbrogates Leukotoxic Activity.

Before proceeding with the in vivo experiments, the inhibitory effect ofthe peptide P17 on the leukotoxic activity of in vitro cultures of M.haemolytica was determined. M. haemolytica cultures were incubated with2, 4, or 5 mg of peptide P17 or the control peptide PSC, and theleukotoxic activity in the culture supernatant fluids was determined bythe dye-reduction cytotoxicity assay. The supernatant fluids from M.haemolytica cultures incubated with the peptide P17 did not exhibitsignificant leukotoxic activity whereas those from cultures incubatedwith the control peptides had significant leukotoxic activity (FIG. 9).The difference in % cytotoxicity exhibited by the supernatant fluidsfrom the cultures incubated with the peptide P17 and PSC wasstatistically significant (P<0.001). Inhibition of cytotoxicity did notdecrease when the quantity of peptide P17 was reduced from 5 to 4 and 2mg, indicating that the peptide P17 can inhibit the Lkt-inducedcytolysis of target cells even at 2 mg (per 5×10⁹ CFU of M.haemolytica).

Pre-Inoculation Status of Calves (Nasopharyngeal Flora and Anti-LktAntibodies).

The results of bacterial isolation are summarized in Table 4. Ten out of12 calves carried M. haemolytica in their pharynx whereas only 4 calvescarried it in the nasal cavity. None of the isolates belonged toserotype 1, the serotype of M. haemolytica used for inoculation. Theunavailability of antisera specific for all known serotypes of M.haemolytica prevented us from identifying the precise serotype of thesebacteria. Pasteurella multocida and Bibersternia trehalosi were isolatedfrom two and three calves, respectively. All the calves used in theexperiment had low titers of Lkt-neutralizing antibodies as revealed bythe cytotoxicity inhibition assay. The titers ranged from 1/20 to 1/320.

TABLE 4 Bacteria isolated from calves pre-inoculation and at necropsy.M. haemolytica P. multocida B. trehalosi Animal # Region Before AfterBefore After Before After 16 P (+) (+) (−) (−) (−) (−) N (−) (+) (−) (−)(−) (−) 21 P (+) (+) (−) (−) (−) (−) N (−) (+) (−) (−) (−) (−) 25 P (+)(+) (−) (−) (−) (−) N (−) (−) (−) (+) (−) (−) 26 P (−) (−) (−) (−) (−)(−) N (−) (−) (−) (+) (−) (−) 29 P (+) (+) (−) (−) (−) (−) N (+) (−) (−)(+) (−) (−) 36 P (+) (+) (−) Past (−) (−) N (−) (−) (+) (+) (−) (−) 52 P(+) (−) (+) (+) (−) (+) N (−) (−) (−) (+) (−) (−) 68 P (+) (−) (−) (+)(−) (−) N (−) (−) (−) (+) (−) (−) 86 P (+) (−) (−) (−) (−) (−) N (−) (−)(−) (+) (−) (−) 146 P (+) (−) (−) (−) (+) (−) N (+) (+) (−) (−) (−) (−)168 P (+) (+) (−) (−) (+) (+) N (+) (+) (−) (−) (−) (−) 170 P (−) (−)(−) (−) (+) (+) N (+) (+) (−) (+) (−) (−) Before: before the challenge;After: at necropsy; P: Pharynx; N: Nasal cavity; (+): Present; (−):Absent; Past: Pasteurella species

Clinical Disease Scores.

Physical examination of each calf was conducted immediately prior toexperimental infection and 6, 18, 24, 42, 48, 66, 72, and 90 hourspost-infection. All calves were clinically normal (clinical score=0)pre-inoculation. Within 6 hours of infection, all calves developedclinical symptoms of disease. Rectal temperature increased to 105-106°F. within 6 hours post-inoculation and returned to baseline 24 hourspost-inoculation as previously reported by other workers (Corrigan etal., 2007). All calves in Groups I and II had nasal discharge throughoutthe study period, but the Group III calves had nasal discharge only upto 24 hours post-inoculation. The clinical scores for nasal discharge ofGroup III calves were statistically significantly different from thoseof Group I and II (P<0.05) of calves inoculated with peptide P17. Thetotal observational disease scores for Group III calves (peptide P17)were lower than those for group I and II at all time points except 48hours. This difference was statistically significant (P<0.05) at 24hours post-inoculation.

Gross Lesions.

All the calves presented gross pulmonary lesions consistent withfibrinonecrotic pneumonia characteristic of M haemolytica-causedpneumonia. The pulmonary lesions in all calves were qualitativelysimilar but differed in severity and extent. Affected lung tissueexhibited consolidation, congestion, and prominent interlobular septaedue to fibrin deposition. These lesions were mainly present in the rightlung and to a limited extent in the left lung (FIG. 11A). The percentvolume of the lungs exhibiting gross pneumonic lesions, as determined bymorphometic techniques (Malazdrewich, et al., 2004) are shown in Table5. The difference in the percent volume of lungs exhibiting grosspneumonic lesions among the three groups was not statisticallysignificant (P=0.9).

Histopathological examination of pulmonary tissues from the calvesrevealed that the lesions observed in the calves were characteristic ofpneumonia caused by M. haemolytica. Interlobular septa were markedlywidened by fibrin and fribrous tissue. Within lobules, discreet foci ofparenchymal necrosis were outlined by dense bands of degenerateneutrophils, often with streaming nuclei Coat cells′). Within necroticfoci, alveolar walls were lysed and alveolar spaces were filled withfibrin, red blood cells and nuclear debris. In some calves largecolonies of coccobacilli were present within affected areas. Parenchymaadjacent to necrotic foci was either collapsed or filled with fibrin andmacrophages (FIG. 11B).

TABLE 5 Gross pneumonic lesions expressed as a % of total lung volumeGroup Animal # Age (months) Lesion (%) Mean (%) I 25 4 16.62 16 4 14.0352 6 3.07 {close oversize brace} 10.03 ± 2.85 146 3 8.38 II 29 4 24.5721 4 4.07 68 6 10.70 {close oversize brace} 12.09 ± 4.39 168 3 9.01 III36 4 15.67 26 4 2.7 86 6 13.22 {close oversize brace} 10.53 ± 3.02 170 38.51Re-Isolation of M. haemolytica Serotype 1 from Pneumonic Lungs ofCalves.

Pure cultures of bacteria (M. haemolytica) were recovered from thepneumonic lungs of all calves. All isolates were identified as M.haemolytica by PCR, and confirmed as serotype 1, by serotyping analysis.All heart blood cultures were negative for M. haemolytica, indicatingthat the infection was confined to the respiratory tract. M. haemolyticawere isolated from pharynx and nasal cavity of most of the calves (Table2). All the isolates from pharynx belonged to serotype 1 but theisolates from nasal cavity were not. Isolation of M. haemolytica fromthe lungs revealed that the calves in Group III carried approximately100- to 1000-fold less organisms in the lungs than the calves in Group Iand Group II (Table 6). This difference, expressed as CFU of M.haemolytica per g of lung tissue, was statistically significant(P<0.001).

TABLE 6 Number of M. haemolytica (CPU per gram of lung tissue) isolatedfrom the lungs of calves at necropsy Group I Group II Group III 2.38 ×10⁶ 1.89 × 10⁷ 5.29 × 10³ 1.50 × 10⁵ 6.73 × 10⁵ 4.92 × 10² 2.68 × 10⁵6.50 × 10⁷ 5.70 × 10⁴  2.9 × 10⁷ 1.87 × 10⁷ 8.00 × 10² Mean 7.9 ± 7.0 ×10⁶ 2.58 ± 1.374 × 10⁷ 1.589 ± 1.374 × 10⁴

Example Discussion

M. haemolytica Lkt-induced cytolysis and degranulation of macrophagesand PMNs is responsible for the acute inflammation and lung injury thatis characteristic of pneumonia caused by this organism. According tocertain embodiments, abrogation of Lkt-induced cytolysis prevents ormitigates the lung lesion. Applicants have shown that a peptiderepresenting the amino acid sequence of Lkt-binding site on itsreceptor, CD18, effectively inhibits the Lkt-induced cytolysis of targetcells (Shanthalingam and Srikumaran, 2009). Applicants have confirmedthat this peptide analog of CD18 abrogates the leukotoxic activity of invitro cultures of M. haemolytica (FIG. 9) which prompted us to test theefficacy of this peptide in a calf challenge model.

M. haemolytica serotype 1 was the obvious choice for this study since itis the serotype that predominantly causes pneumonia in cattle althoughother serotypes such as 2, 4 and 7 commonly inhabits the nasopharynx ofhealthy cattle (Frank and Smith 1983, Frank, 1988; Gonzalez andMaheswaran, 1993). All the pharyngeal isolates obtained from the calvespre-inoculation belonged to serotypes other than serotype 1 allowed usto track the inoculated M. haemolytica by serotyping. The presence ofLkt-neutralizing antibodies in the serum indicates that the calves hadthese antibodies in the epithelial lining fluid which, according tocertain embodiments, had an effect on the bacterial clearance. Thecalves were matched for Lkt-neutralizing antibody titers. According toadditional aspects, colostrum-deprived calves are used to eliminate anypossible effects due to Lkt-neutralizing antibodies.

All the calves developed high rectal temperature within 6 hours ofinoculation which dropped to normal levels in 24 hours. LPS represents10 to 25% of the dry weight of M. haemolytica bacteria (Keiss et al.,1964) and it forms high-molecular-weight aggregates with Lkt (Li andClinckenbeard, 1999). Since LPS stimulates alveolar macrophages toproduce TNFα and interleukin-8, leading to inflammation, it is likelythat some of the effects that we observed were LPS related. The totalobservational disease scores for Group III calves were lower than thosefor group I and II at all time points beyond 6 hours. This differencewas statistically significant (P<0.05) at 24 hours post-inoculation.According to certain embodiments, the peptide is absorbed from thesurface of the respiratory epithelium by 24 hours post-inoculation,which results in the loss of protective effect of the peptide. Accordingto additional aspects, use of peptides absorbed to solid particles whichslowly release the peptide provides for prolonging the protective effectof the peptides which mitigate the disease.

The peptide P17 strongly inhibited Lkt-induced cytolysis of bovine PMNsin in vitro assays. It is surprising that the gross pneumonic lesions incalves inoculated with this peptide and M. haemolytica (Group III) wereno less than those in calves inoculated with the control peptide PSC andM. haemolytica (Group II), or M. haemolytica alone (Group I). Accordingto particular aspects, the ability of the peptide to mitigate lunglesions could be enhanced by: (1) increasing the quantity of thepeptide; (2) decreasing absorption of the peptide from the lungepithelial surface; or (3) protecting the peptide from proteolyticdegradation. For example, by use of peptides adsorbed on solid particleswhich slowly release the peptide is likely to prolong the protectiveeffect of the peptides which could be expected to mitigate the lunglesions (Freiberg and Zhu, 2004; Schroder and Stahl, 1984; Schroder,1985). Although the lung lesions were similar in extent in all threegroups, it is possible that the animals in Group III would haverecovered from the disease if they were not euthanized at 90 hourspost-inoculation, as we did in this study. This scenario is supported bythe finding that the number of M. haemolytica recovered from the lungsof Group III animals was 100 to 1000 times less than that recovered fromthe lungs of animals in Group I and II (Table 6). The significantlylower number of bacteria isolated from the lungs of Group III animals islikely due to the presence of relatively larger number of functionalphagocytes in the lungs which were protected from Lkt by the peptides.In contrast, the animals in Groups I and II would have had relativelysmaller number of phagocytes in the lungs because of their cytolysis byLkt.

Molecules such as proteins and peptides are often marginally stable andconsequently could be easily damaged or degraded (Tibbetts et al.,2000). In vivo degradation when exposed to enzymes results in shortbiological half-lives (Tibetts, 2000). According to particular aspects,prolonging the presence of peptides in the lungs extends protection ofthe phagocytes from the Lkt, resulting in more effective clearance ofbacteria from the lungs which in turn prevents or mitigates lunglesions.

In particular aspects, nanoparticle delivery systems are used to improveprotein/peptide stability and provide sustained release. Adsorbing thepeptides to solid particles such as dextran represents a method ofprolonging the presence and activity of the peptides in the lungs.Dextran, under certain controlled conditions, aggregates into porousmicrospheres, forming crystallized dextran microspheres that canabsorb/adsorb peptides, drugs and biologicals, protecting them againstdegradation and prolonging their release. Such dextran microspheres areavailable which are biodegradable, biocompatible, non-toxic,non-immunogenic and are removed from the body by normal physiologicalroutes. These characteristics are uniquely advantageous for peptidedelivery. According to particular aspects, crystallized dextranmicrospheres are used for the delivery of peptides and prolong theprotective effects of the peptide.

References cited for Examples 1-6, and incorporated herein by referencefor their relevant teachings as referred to herein:

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1-38. (canceled)
 39. A genetically engineered ruminant animal,comprising a ruminant cell that is genetically engineered to contain anucleic acid sequence encoding a ruminant CD18 protein having acleavable signal peptide with a helix-breaking amino acid residue an atamino acid positioned 5 residues upstream of the signal peptide cleavagesite, wherein said nucleic acid sequence is incorporated into the genomeof the genetically engineered ruminant animal.
 40. The geneticallyengineered ruminant animal of claim 39, wherein said animal is a cow.41. The genetically engineered ruminant animal of claim 39, wherein theamino acid residue at amino acid position 18 is selected from the groupconsisting of glycine, proline, arginine, and tyrosine.
 42. A method ofproviding a genetically engineered ruminant animal, comprisingintroduction into, or engineering within one or more cells of a ruminantanimal, a nucleic acid sequence encoding a ruminant CD18 protein havinga cleavable signal peptide with a helix-breaking amino acid residue atamino acid positioned 5 residues upstream of the signal peptide cleavagesite, wherein the genetically engineered ruminant animal is lesssusceptible to, or resistant to the effects of M. haemolytica, relativeto wild-type control cells.
 43. The method of claim 42, wherein saidanimal is a cow.
 44. The method of claim 42, wherein the amino acidresidue at amino acid position 18 is selected from the group consistingof glycine, proline, arginine, and tyrosine.